Compositions against cat allergy

ABSTRACT

The present invention relates to the use of a composition in a method of reducing the allergenicity of a cat. Moreover, the present invention relates to the use of a composition in a method of reducing the allergenicity of a cat for a human exposed to the cat. Furthermore, the present invention relates to compositions comprising a virus-like particle (VLP) and at least one Fel d1 protein. The compositions of the invention induce efficient immune responses, in particular antibody responses, in cats and are useful for the treatment and/or prevention of cat allergy.

The present invention relates to the use of a composition in a method ofreducing the allergenicity of a cat for a human. Moreover, the presentinvention relates to the use of a composition in a method of reducingthe allergenicity of a cat for a human exposed to the cat. Furthermore,the present invention relates to compositions comprising a virus-likeparticle (VLP) and at least one Fel d1 protein. The compositions of theinvention induce efficient immune responses, in particular antibodyresponses, in cats reducing the allergenicity of the Fel d1 shed by thecats and are, therefore, useful for the treatment and/or prevention ofcat allergy in humans.

RELATED ART

The domestic cat (Felis domesticus) is an important source of indoorallergens (Lau, S., et al. (2000) Lancet 356, 1392-1397). Indeed, catsare found in about 25% of households in Western countries and allergy tocats is found in a large part of the population. The severity ofsymptoms range from relatively mild rhinitis and conjunctivitis topotentially life-threatening asthmatic exacerbation. Although patientsare occasionally sensitized to several different molecules in cat danderand pelts, the major allergen is Fel d1. The importance of this allergenhas been emphasized in numerous studies. In fact more than 80% of catallergic patients exhibit IgE antibodies to this potent allergen (vanRee, R., et al. (1999) J. Allergy Clin Immunol 104, 1223-1230).

Fel d1 is a 35-39 kDa acidic glycoprotein containing 10-20% N-linkedcarbohydrates and is found in the pelt, i.e. the skin and the fur, inthe salivary and lacrimal glands as well as in perianal glands of cats.It is formed by two non-covalently linked heterodimers. Each heterodimerconsists of one 70 residue peptide (known as “chain 1”) and one 78, 85,90 or 92 residue peptide (known as “chain 2”) which are encoded byseparate genes (see Duffort, O. A., et al. (1991) Mol Immunol 28,301-309; Morgenstern, J. P., et al; (1991) Proc Natl Acad Sci USA 88,9690-9694 and Griffith, I. J., et al. (1992) Gene 113, 263-268).

Treatment of cat allergic patients is currently effected bydesensitization therapy involving repeated injections with increasingdosages of either a crude cat dander extract or short peptides derivedfrom Fel d1. Lilja et al and Hedlin et al have disclosed adesensitization program in the course of which crude cat dander extractshave been given to cat allergic patients (Lilja, Q, et al. (1989) JAllergy Clin Immunol 83, 37-44 and Hedlin, et al. (1991) J Allergy ClinImmunol 87, 955-964). This program took at least two to three years andthe patients after three year treatment still had systemic symptoms.Using short peptides derived from Fel d1 for desensitization resulted innon-significant difference between the peptide group and the placebogroup (Oldfield, W. L., et al. (2002) Lancet 360, 47-53). Efficacy wasonly seen when large amount (750 μg) of the short peptide was given topatients (Norman, P. S., et al. (1996) Am J Respir Crit Care Med 154,1623-1628).

Allergic side effects, such as late asthmatic reactions, have beenreported in both crude cat dander extract treatment and in short peptidetreatment. Therefore, anaphylactic shock due to the injected allergen isof great safety concern for any desensitization program. Avoidance ofsuch effect by reducing the injected amount of allergen, however, eitherreduces the efficacy of the treatment or prolongs the treatment. Thus,there is a great need in the field of cat-allergy treatment foralternative desensitization regimes, and hereby in particular fordesensitization regimes that are able to reduce allergic symptoms, butdo not trigger allergic side reaction. Active immunization in humanswith Fel d1 antigens covalently linked to virus-like particles has alsobeen described to address cat allergy in humans (WO2006/097530A2).

Alternatively, treating the cat itself has been suggested to reduce theamount of Fel d1 shed by a cat (WO2007/113633A2). However, no data, letalone reports of success, have ever since been provided.

As a consequence, there is a need for compositions and treatments shownto be effective in addressing cat allergy in humans. In particular,there is a need for compositions and treatments shown to be effective ina method of reducing the allergenicity of a cat for a human.

SUMMARY OF THE INVENTION

We have shown that compositions of the present invention are effectivein a method of reducing the allergenicity of a cat, and hereby inparticular the allergenicity of a cat for a human. Thus, we have foundthat administration of the compositions of the present invention to acat led to the generation of Fel d1-specific IgG antibodies as well asof Fel d1-specific IgA antibodies. Moreover, immune complexes consistingof endogenous Fel d1 and IgA antibodies were detected in the immunizedcats. Furthermore, saliva extracts from cats taken after immunizationwith the said compositions showed decreased levels of degranulation ofbasophils from cat allergic patients by up to 20% when compared tosaliva extracts taken from said cats before immunization whichcorresponds to a 13-fold decrease in Fel d1 concentration and indicatingthat a significant reduction in allergenic Fel d1 in saliva wasachieved.

Without being bound by this explanation, the present invention impactsthe allergic response in humans at the first possible point ofintervention by inducing Fel d1-specific IgG and IgA antibodies in cats,which will bind Fel d1 and thus lower or neutralize the allergeniceffect of Fel d1. Upon administration of an effective amount of thecompositions of the present invention, a humoral immune response againstFel d1 as well against the VLP carrier is induced in the cat. Theantibody response is expected to be predominantly of the IgG isotype butalso IgA will be induced. These anti-Fel d1 antibodies eventuallymediate protection from the allergic reaction. Following immunizationand induction of Fel d1-specific antibodies, immune complexes, i.e.antibody-Fel d1 complexes, will form in situ and be secreted into theenvironment. Consequently humans will be exposed to complexed Fel d1 andless of the natural unbound (“reactive”) form shed by the cat. This islikely to be effective via two mechanisms of action. First by reducingthe engagement of Fel d1 by IgE/FcεRI (classical neutralization) andsecond through co-engagement of IgE/FcεRI and IgG/FcγRIIb which cande-activate FcεRI mediated signaling (negative signaling).

Thus, in a first aspect, the present invention provides for use of acomposition in a method of reducing the allergenicity of a cat typicallyand preferably for a human, wherein an effective amount of saidcomposition is administered to said cat, and wherein said compositioncomprises (i) a virus-like particle with at least one first attachmentsite; (ii) at least one Fel d1 protein with at least one secondattachment site; and wherein said virus-like particle and said Fel d1protein are linked through said at least one first and said at least onesecond attachment site. Preferably, said method is a non-therapeuticmethod of reducing the allergenicity of said cat. In a furtherembodiment, said cat is not suffering from an allergy or an auto-immunedisease, typically and preferably wherein said cat is not suffering froman allergy or an auto-immune disease caused by Fel d1.

In a preferred embodiment, said reducing the allergenicity of said cat,typically and preferably for a human, is effected by generating immunecomplexes formed of Fel d1 and Fel d1-antibodies in the saliva, the fur,the skin or the tears of said cat, preferably in the saliva of said cat,and wherein preferably said administration of said composition leads tosaid generating of said immune complexes in the saliva, fur, skin ortears of said cat, preferably in the saliva of said cat.

In a further preferred embodiment, said VLP is a modified VLP ofcucumber mosaic virus (CMV), wherein said modified VLP of CMV comprises,essentially consists of, or alternatively consists of, at least onemodified CMV polypeptide, wherein said modified CMV polypeptidecomprises, or preferably consists of, (a) a CMV polypeptide, and (b) a Thelper cell epitope; and wherein said CMV polypeptide comprises, orpreferably consists of, (i) an amino acid sequence of a coat protein ofCMV; or (ii) a mutated amino acid sequence, wherein the amino acidsequence to be mutated is an amino acid sequence of a coat protein ofCMV, and wherein said mutated amino acid sequence and said coat proteinof CMV show a sequence identity of at least 90%, preferably of at least95%, further preferably of at least 98% and again more preferably of atleast 99%. In a further very preferred embodiment, said CMV polypeptidecomprises, or preferably consists of, (a) an amino acid sequence of acoat protein of CMV, wherein said amino acid sequence comprises, orpreferably consists of, SEQ ID NO: 1 or (b) an amino acid sequencehaving a sequence identity of at least 90% of SEQ ID NO:1; and whereinsaid amino sequence as defined in (a) or (b) in this claim comprises SEQID NO:34; or wherein said amino sequence as defined in (a) or (b) inthis claim comprises an amino acid sequence region, wherein said aminoacid sequence region has a sequence identity of at least 90% with SEQ IDNO:34.

In a further very preferred embodiment, said modified CMV polypeptidecomprises, preferably consists of, an amino acid sequence of SEQ ID NO:6or SEQ ID NO:7. Furthermore, very preferably said Fel d1 protein is aFel d1 fusion protein comprising chain 1 of Fel d1 and chain 2 of Feld1, wherein chain 1 of Fel d1 and chain 2 of Fel d1 are fused eitherdirectly via one peptide bond or via a spacer, which links theN-terminus of one chain with the C-terminus of another chain. Verypreferably, said Fel d1 protein comprises an amino acid sequenceselected from: (a) SEQ ID NO:20; (b) SEQ ID NO:25; (c) SEQ ID NO:26; (d)SEQ ID NO:27; or (e) SEQ ID NO:29.

In another aspect, the present invention provides for a method forreducing the allergenicity of a cat typically and preferably for ahuman, wherein said method comprises administering an effective amountof said composition to said cat, wherein said composition comprises (i)a virus-like particle with at least one first attachment site; (ii) atleast one Fel d1 protein with at least one second attachment site; andwherein said virus-like particle and said Fel d1 protein are linkedthrough said at least one first and said at least one second attachmentsite. Preferably, said method is a non-therapeutic method of reducingthe allergenicity of said cat.

In a further aspect, the present invention provides for a compositioncomprising (i) a virus-like particle (VLP) with at least one firstattachment site; (ii) at least one Fel d1 protein with at least onesecond attachment site; and wherein said virus-like particle and saidFel d1 protein are linked through said at least one first and said atleast one second attachment site, and wherein said Fel d1 proteincomprises an amino acid sequence selected from SEQ ID NO:25 or SEQ IDNO:27; and wherein said VLP is a modified VLP of cucumber mosaic virus(CMV), wherein said modified VLP of CMV comprises, essentially consistsof, or alternatively consists of, at least one modified CMV polypeptide,wherein said modified CMV polypeptide comprises, or preferably consistsof, (a) a CMV polypeptide, and (b) a T helper cell epitope; and whereinsaid modified CMV polypeptide comprises, preferably consists of, anamino acid sequence of SEQ ID NO:6 or SEQ ID NO:7.

In a further aspect, the present invention provides an immunogeniccomposition formulated as a composition for administration to the catthat reduces the allergenicity of the Fel d1 shed by the cat. Thecomposition of the invention renders the cat less allergenic towardshumans prone to show symptoms of cat allergy when in contact with orproximity to the cat.

In another aspect, the present invention provides for use of acomposition in a method of reducing the allergenicity of a cat, whereinan effective amount of said composition is administered to said cat, andwherein said composition comprises (i) a virus-like particle with atleast one first attachment site; (ii) at least one Fel d1 protein withat least one second attachment site; and wherein said virus-likeparticle and said Fel d1 protein are linked through said at least onefirst and said at least one second attachment site, and wherein saidreduced allergenicity of said cat is a reduced allergenicity of said catfor a human.

In another aspect, the present invention provides for a composition foruse in a method of reducing the allergenicity of a cat, wherein aneffective amount of said composition is administered to said cat, andwherein said composition comprises (i) a virus-like particle with atleast one first attachment site; (ii) at least one Fel d1 protein withat least one second attachment site; and wherein said virus-likeparticle and said Fel d1 protein are linked through said at least onefirst and said at least one second attachment site, and wherein saidreduced allergenicity of said cat is a reduced allergenicity of said catfor a human

Further aspects and embodiments of the present invention will be becomeapparent as this description continues.

BRIEF DESCRIPTION OF FIGURES

FIG. 1 pET-CMVwt plasmid map. The relative positions of relevant genesand restriction enzyme sites are denoted.

FIG. 2A Dynamic light scattering of purified CMVwt VLPs. The size ofparticles was detected by using Zetasizer Nano ZS (Malvern InstrumentsLtd., United Kingdom).

FIG. 2B Electron-microscopy analysis of purified CMVwt VLPs. For themorphological analysis of VLPs the JEM-1230 electron microscope (JeolLtd., Tokyo, Japan) was used.

FIG. 3 Mass spectrometric analysis of purified CMV-derived VLPs.Matrix-assisted laser desorption/ionization (MALDI)-TOF MS analysis wascarried out on an Autoflex MS (Bruker Daltonik, Germany). The proteinmolecular mass (MM) calibration standard II (22.3-66.5 kDa; BrukerDaltonik) was used for mass determination.

FIG. 3A CMVwild-type (“wt”); theoretical MM=24069; found MM=24058

FIG. 3B CMV-Npadr; theoretical MM=24161 (without first Met); foundMM=24160

FIG. 3C CMV-Ntt830; theoretical MM=24483 (without first Met); foundMM=24477

FIG. 4A Dynamic light scattering of purified CMV-Ntt830 VLPs. The sizeof particles was detected by using Zetasizer Nano ZS (MalvernInstruments Ltd., United Kingdom).

FIG. 4B Electron-microscopy analysis of purified CMV-Ntt830 VLPs. Forthe morphological analysis of VLPs the JEM-1230 electron microscope(Jeol Ltd., Tokyo, Japan) was used.

FIG. 5A Dynamic light scattering of purified CMV-Npadr VLPs. The size ofparticles was detected by using Zetasizer Nano ZS (Malvern InstrumentsLtd., United Kingdom).

FIG. 5B Electron-microscopy analysis of purified CMV-Npadr VLPs. For themorphological analysis of VLPs the JEM-1230 electron microscope (JeolLtd., Tokyo, Japan) was used.

FIG. 6A SDS/PAGE analysis of expression and purification of F12H6GGCprotein from E. coli C2566 cells, using PrepEase kit (USB). M—proteinsize marker; S—soluble protein fraction; P—cell debris; F—Flow throughfrom Ni-IDA column (unbound proteins); W1, W2—Wash fractions (2×2 ml1×LEW buffer) W3, W4 Wash fractions (2×2 ml 1×LEW+10 mM imidazole); E1,E2—Elution fractions (2×1.5 ml E buffer 250 mM imidazole).

FIG. 6B Mass spectrometric analysis of purified F12H6GGC. The calculatedaverage mass of the F12H6GGC corresponds to 20089.8 Da. The observedmass of 20105.3 corresponds to F12H6GGC with one Met sulfoxide.

FIG. 6C Coomassie Blue stained SDS-PAGE analysis of purification ofFG12GGCG. (A) s—post sonication supernatant; AmS—dissolved precipitateafter 50% (NH₄)₂SO₄. Various fractions from the DEAE column procedure:FT—flow through, A4-A7—fractions eluted by increasing NaCl gradient (B)Subsequent purification by MonoQ and Butyl HP columns.

FIG. 7 A sandwich ELISA supplied from Indoor Biotechnologies using mAbsraised against the natural Fel d1 is shown. The mAbs recognize F12H6GGCand natural Fel d1 equally well.

FIG. 8A Basophil activation test (BAT) for natural Fel d1.

FIG. 8B Basophil activation test (BAT) for F12H6GGC. F12H6GGC andnatural Fel d1 induce similar activation levels of basophils in bloodfrom cat allergic patients indicated by the up-regulation of CD63 onCCR3+ basophils.

FIG. 9 Antibody response of mice which received 10 μg of either Feld1-CMV VLPs (Fel d1-CMV-Ntt830-VLP or Fel d1-CMV-Npadr-VLP) or CMV-VLPs(CMV-Ntt830-VLP or CMV-Npadr-VLP) simply mixed with Fel d1 fusionprotein F12H6GGC on day 0 and day 14. Serum was collected on day 0, 14and 21 and analyzed by ELISA for natural Fel d1 specific IgG-antibodies.N=3.

FIG. 10 IgG-antibody titer against Fel d1 and CMV in cats immunized withFel d1-CMV-Ntt830-VLP with or without adjuvant. ELISAs were used todetect Fel d1- (FIG. 10A) and CMV- (FIG. 10B) specific IgG antibodies insera from immunized cats.

FIG. 11 Measurement of anti-Fel d1 and anti-CMV antibodies in salivaextracts of cats. ELISAs were used to detect Fel d1-specific IgGantibodies (FIG. 11A), Fel d1-specific IgA antibodies (FIG. 11B),CMV-specific IgG antibodies (FIG. 11C) and CMV-specific IgA antibodies(FIG. 11D).

FIG. 12 Detection of immune complexes consisting of endogenous Fel d1and IgA antibodies in saliva of immunized cats.

FIG. 13 Basophil activation test (BAT) with saliva samples from day 0and day 85 show immunization with Fel d1-CMV-Ntt830-VLP reducesdegranulation in 5 of 6 cats (FIG. 13A and FIG. 13B).

FIG. 14 Comparison of wheal size (area, mm2) from skin prick tests usingcat fur extract obtained before and after immunization with Feld1-CMV-Ntt830-VLP. Data, mean+/−standard error of the mean, are shownfor cat fur extracts diluted 1:80 and 1:243 (1:240). A total of 16 skinprick tests comparing wheal size with pre and post-immunization furextracts were successfully performed and analyzed.

DETAILED DESCRIPTION OF THE INVENTION

Unless defined otherwise, all technical and scientific terms used hereinhave the same meanings as commonly understood by one of ordinary skillin the art to which this invention belongs.

Virus-like particle (VLP): The term “virus-like particle (VLP)” as usedherein, refers to a non-replicative or non-infectious, preferably anon-replicative and non-infectious virus particle, or refers to anon-replicative or non-infectious, preferably a non-replicative andnon-infectious structure resembling a virus particle, preferably acapsid of a virus. The term “non-replicative”, as used herein, refers tobeing incapable of replicating the genome comprised by the VLP. The term“non-infectious”, as used herein, refers to being incapable of enteringthe host cell. A virus-like particle in accordance with the invention isnon-replicative and non-infectious since it lacks all or part of theviral genome or genome function. A virus-like particle in accordancewith the invention may contain nucleic acid distinct from their genome.Recombinantly produced virus-like particles typically contain host cellderived RNA. A typical and preferred embodiment of a virus-like particlein accordance with the present invention is a viral capsid composed ofpolypeptides of the invention. A virus-like particle is typically amacromolecular assembly composed of viral coat protein which typicallycomprises 60, 120, 180, 240, 300, 360, or more than 360 protein subunitsper virus-like particle. Typically and preferably, the interactions ofthese subunits lead to the formation of viral capsid or viral-capsidlike structure with an inherent repetitive organization. One feature ofa virus-like particle is its highly ordered and repetitive arrangementof its subunits.

Virus-like particle of CMV: The terms “virus-like particle of CMV” orCMV VLPs refer to a virus-like particle comprising, or preferablyconsisting essentially of, or preferably consisting of at least one CMVpolypeptide. Preferably, a virus-like particle of CMV comprises said CMVpolypeptide as the major, and even more preferably as the sole proteincomponent of the capsid structure. Typically and preferably, virus-likeparticles of CMV resemble the structure of the capsid of CMV. Virus-likeparticles of CMV are non-replicative and/or non-infectious, and lack atleast the gene or genes encoding for the replication machinery of theCMV, and typically also lack the gene or genes encoding the protein orproteins responsible for viral attachment to or entry into the host.This definition includes also virus-like particles in which theaforementioned gene or genes are still present but inactive. Preferredmethods to render a virus-like particle of CMV non replicative and/ornon-infectious is by physical or chemical inactivation, such as UVirradiation, formaldehyde treatment. Preferably, VLPs of CMV lack thegene or genes encoding for the replication machinery of the CMV, andalso lack the gene or genes encoding the protein or proteins responsiblefor viral attachment to or entry into the host. Again more preferably,non-replicative and/or non-infectious virus-like particles are obtainedby recombinant gene technology. Recombinantly produced virus-likeparticles of CMV according to the invention typically and preferably donot comprise the viral genome. Virus-like particles comprising more thanone species of polypeptides, often referred to as mosaic VLPs are alsoencompassed by the invention. Thus, in one embodiment, the virus-likeparticle according to the invention comprises at least two differentspecies of polypeptides, wherein at least one of said species ofpolypeptides is a CMV polypeptide. Preferably, a VLP of CMV is amacromolecular assembly composed of CMV coat protein which typicallycomprises 180 coat protein subunits per VLP. Typically and preferably, aVLP of CMV as used herein, comprises, essentially consists of, oralternatively consists of, at least one CMV polypeptide comprising orpreferably consisting of (i) an amino acid sequence of a coat protein ofCMV; or (ii) a mutated amino acid sequence, wherein the amino acidsequence to be mutated is an amino acid sequence of a coat protein ofCMV, and wherein said mutated amino acid sequence and said amino acidsequence to be mutated show a sequence identity of at least 90%,preferably of at least 95%, further preferably of at least 98% and againmore preferably of at least 99%.

Polypeptide: The term “polypeptide” as used herein refers to a polymercomposed of amino acid monomers which are linearly linked by peptidebonds (also known as amide bonds). The term polypeptide refers to aconsecutive chain of amino acids and does not refer to a specific lengthof the product. Thus, peptides, and proteins are included within thedefinition of polypeptide.

Cucumber Mosaic Virus (CMV) polypeptide: The term “cucumber mosaic virus(CMV) polypeptide” as used herein refers to a polypeptide comprising orpreferably consisting of: (i) an amino acid sequence of a coat proteinof cucumber mosaic virus (CMV), or (ii) a mutated amino acid sequence,wherein the amino acid sequence to be mutated is an amino acid sequenceof a coat protein of CMV, and wherein said mutated amino acid sequenceand said amino acid sequence to be mutated, i.e. said coat protein ofCMV, show a sequence identity of at least 90%, preferably of at least95%, further preferably of at least 98% and again more preferably of atleast 99%. Typically and preferably, the CMV polypeptide is capable offorming a virus-like particle of CMV upon expression by self-assembly.

Coat protein (CP) of cucumber mosaic virus (CMV): The term “coat protein(CP) of cucumber mosaic virus (CMV)”, as used herein, refers to a coatprotein of the cucumber mosaic virus which occurs in nature. Due toextremely wide host range of the cucumber mosaic virus, a lot ofdifferent strains and isolates of CMV are known and the sequences of thecoat proteins of said strains and isolates have been determined and are,thus, known to the skilled person in the art as well. The sequences ofsaid coat proteins (CPs) of CMV are described in and retrievable fromthe known databases such as Genbank, dpvweb.net orncbi.nlm.nih.gov/protein/. Examples are described in EP Application No.14189897.3. Further examples of CMV coat proteins are provided in SEQ IDNOs 1-3. It is noteworthy that these strains and isolates have highlysimilar coat protein sequences at different protein domains, includingthe N-terminus of the coat protein. In particular, 98.1% of allcompletely sequenced CMV isolates share more than 85% sequence identitywithin the first 28 amino acids of their coat protein sequence, andstill 79.5% of all completely sequenced CMV isolates share more than 90%sequence identity within the first 28 amino acids of their coat proteinsequence.

Typically and preferably, the coat protein of CMV used for the presentinvention is capable of forming a virus-like particle of CMV uponexpression by self-assembly. Preferably, the coat protein of CMV usedfor the present invention is capable of forming a virus-like particle ofCMV upon expression by self-assembly in E. coli.

Modified virus-like particle (VLP) of cucumber mosaic virus (CMV): Theterm “modified virus-like particle (VLP) of cucumber mosaic virus (CMV)”as used herein, refers to a VLP of CMV which is a modified one in suchas it comprises, or preferably consists essentially of, or preferablyconsists of at least one modified CMV polypeptide, wherein said modifiedCMV polypeptide comprises, or preferably consists of, a CMV polypeptide,and a T helper cell epitope. Typically and preferably, said T helpercell epitope (i) is fused to the N-terminus of said CMV polypeptide,(ii) is fused to the C-terminus of said CMV polypeptide, (iii) replacesa region of consecutive amino acids of said CMV polypeptide, wherein thesequence identity between said replaced region of consecutive aminoacids of said CMV polypeptide and the T helper cell epitope is at least15%, preferably at least 20%, or (iv) replaces a N-terminal region ofsaid CMV polypeptide, and wherein said replaced N-terminal region ofsaid CMV polypeptide consists of 5 to 15 consecutive amino acids.Preferably, said T helper cell epitope replaces a N-terminal region ofsaid CMV polypeptide, and wherein said replaced N-terminal region ofsaid CMV polypeptide consists of 5 to 15 consecutive amino acids,preferably of 9 to 14 consecutive amino acids, more preferably of 11 to13 consecutive amino acids, and most preferably of 11, 12 or 13consecutive amino acids. Preferably said modified VLP of CMV of thepresent invention is a recombinant modified VLP of CMV.

Modified CMV polypeptide: The term “modified CMV polypeptide” as usedherein refers to a CMV polypeptide modified in such as defined herein,that said modified CMV polypeptide comprises, or preferably consists of,a CMV polypeptide, and a T helper cell epitope. Typically, the modifiedCMV polypeptide is capable of forming a virus-like particle of CMV uponexpression by self-assembly. Preferably, the modified CMV polypeptide isa recombinant modified CMV polypeptide and is capable of forming avirus-like particle of CMV upon expression by self-assembly in E. coli.

N-terminal region of the CMV polypeptide: The term “N-terminal region ofthe CMV polypeptide” as used herein, refers either to the N-terminus ofsaid CMV polypeptide, and in particular to the N-terminus of a coatprotein of CMV, or to the region of the N-terminus of said CMVpolypeptide or said coat protein of CMV but starting with the secondamino acid of the N-terminus of said CMV polypeptide or said coatprotein of CMV if said CMV polypeptide or said coat protein comprises aN-terminal methionine residue. Preferably, in case said CMV polypeptideor said coat protein comprises a N-terminal methionine residue, from apractical point of view, the start-codon encoding methionine willusually be deleted and added to the N-terminus of the Th cell epitope.Further preferably, one, two or three additional amino acids, preferablyone amino acid, may be optionally inserted between the statingmethionine and the Th cell epitope for cloning purposes. The term“N-terminal region of the mutated amino acid sequence of a CMVpolypeptide or a CMV coat protein” as used herein, refers either to theN-terminus of said mutated amino acid sequence of said CMV polypeptideor said coat protein of CMV, or to the region of the N-terminus of saidmutated amino acid sequence of said CMV polypeptide or said coat proteinof CMV but starting with the second amino acid of the N-terminus of saidmutated amino acid sequence of said CMV polypeptide or said coat proteinof CMV if said mutated amino acid sequence comprises a N-terminalmethionine residue. Preferably, in case said CMV polypeptide or saidcoat protein comprises a N-terminal methionine residue, from a practicalpoint of view, the start-codon encoding methionine will usually bedeleted and added to the N-terminus of the Th cell epitope. Furtherpreferably, one, two or three additional amino acids, preferably oneamino acid, may be optionally inserted between the stating methionineand the Th cell epitope for cloning purposes.

Recombinant polypeptide: In the context of the invention the term“recombinant polypeptide” refers to a polypeptide which is obtained by aprocess which comprises at least one step of recombinant DNA technology.Typically and preferably, a recombinant polypeptide is produced in aprokaryotic expression system. It is apparent for the artisan thatrecombinantly produced polypeptides which are expressed in a prokaryoticexpression system such as E. coli may comprise an N-terminal methionineresidue. The N-terminal methionine residue is typically cleaved off therecombinant polypeptide in the expression host during the maturation ofthe recombinant polypeptide. However, the cleavage of the N-terminalmethionine may be incomplete. Thus, a preparation of a recombinantpolypeptide may comprise a mixture of otherwise identical polypeptideswith and without an N-terminal methionine residue. Typically andpreferably, a preparation of a recombinant polypeptide comprises lessthan 10%, more preferably less than 5%, and still more preferably lessthan 1% recombinant polypeptide with an N-terminal methionine residue.

Recombinant CMV polypeptide: The term “recombinant CMV polypeptide”refers to a CMV polypeptide as defined above which is obtained by aprocess which comprises at least one step of recombinant DNA technology.Typically and preferably a preparation of a recombinant CMV polypeptidecomprises less than 10%, more preferably less than 5%, and still morepreferably less than 1% recombinant CMV polypeptide with an N-terminalmethionine residue. Consequently, a recombinant virus-like particle ofthe invention may comprise otherwise identical recombinant polypeptideswith and without an N-terminal methionine residue.

Recombinant modified CMV polypeptide: The term “recombinant modified CMVpolypeptide” refers to a modified CMV polypeptide as defined above whichis obtained by a process which comprises at least one step ofrecombinant DNA technology. Typically and preferably a preparation of arecombinant modified CMV polypeptide comprises less than 10%, morepreferably less than 5%, and still more preferably less than 1%recombinant modified CMV polypeptide with an N-terminal methionineresidue. Consequently, a recombinant virus-like particle of theinvention may comprise otherwise identical recombinant polypeptides withand without an N-terminal methionine residue.

Recombinant virus-like particle: In the context of the invention theterm “recombinant virus-like particle” refers to a virus-like particle(VLP) which is obtained by a process which comprises at least one stepof recombinant DNA technology. Typically and preferably, a recombinantvirus-like particle comprises at least one recombinant polypeptide,preferably a recombinant CMV polypeptide or recombinant modified CMVpolypeptide. Most preferably, a recombinant virus-like particle iscomposed of or consists of recombinant CMV polypeptides or recombinantmodified CMV polypeptides. As a consequence, if in the context of thepresent invention the definition of inventive recombinant VLPs areeffected with reference to specific amino acid sequences comprising aN-terminal methionine residue the scope of these inventive recombinantVLPs encompass the VLPs formed by said specific amino acid sequenceswithout said N-terminal methionine residue but as well, even thoughtypically in a minor amount as indicated herein, the VLPs formed by saidspecific amino acid sequences with said N-terminal methionine.Furthermore, it is within the scope of the present invention that if thedefinition of inventive recombinant VLPs are effected with reference tospecific amino acid sequences comprising a N-terminal methionine residueVLPs are encompassed comprising both amino acid sequences comprisingstill said N-terminal methionine residue and amino acid sequenceslacking the N-terminal methionine residue.

Mutated amino acid sequence: The term “mutated amino acid sequence”refers to an amino acid sequence which is obtained by introducing adefined set of mutations into an amino acid sequence to be mutated. Inthe context of the invention, said amino acid sequence to be mutatedtypically and preferably is an amino acid sequence of a coat protein ofCMV. Thus, a mutated amino acid sequence differs from an amino acidsequence of a coat protein of CMV in at least one amino acid residue,wherein said mutated amino acid sequence and said amino acid sequence tobe mutated show a sequence identity of at least 90%. Typically andpreferably said mutated amino acid sequence and said amino acid sequenceto be mutated show a sequence identity of at least 91%, 92%, 93% 94%,95%, 96%, 97%, 98%, or 99%. Preferably, said mutated amino acid sequenceand said sequence to be mutated differ in at most 11, 10, 9, 8, 7, 6, 4,3, 2, or 1 amino acid residues, wherein further preferably saiddifference is selected from insertion, deletion and amino acid exchange.Preferably, the mutated amino acid sequence differs from an amino acidsequence of a coat protein of CMV in least one amino acid, whereinpreferably said difference is an amino acid exchange.

Position corresponding to residues . . . : The position on an amino acidsequence, which is corresponding to given residues of another amino acidsequence can be identified by sequence alignment, typically andpreferably by using the BLASTP algorithm, most preferably using thestandard settings. Typical and preferred standard settings are: expectthreshold: 10; word size: 3; max matches in a query range: 0; matrix:BLOSUM62; gap costs: existence 11, extension 1; compositionaladjustments: conditional compositional score matrix adjustment.

Sequence identity: The sequence identity of two given amino acidsequences is determined based on an alignment of both sequences.Algorithms for the determination of sequence identity are available tothe artisan. Preferably, the sequence identity of two amino acidsequences is determined using publicly available computer homologyprograms such as the “BLAST” program (blast.ncbi.nlm.nih.gov/Blast.cgi)or the “CLUSTALW” (www.genome.ip/tools/clustalw/), and hereby preferablyby the “BLAST” program provided on the NCBI homepage atblast.ncbi.nlm.nih.gov/Blast.cgi, using the default settings providedtherein. Typical and preferred standard settings are: expect threshold:10; word size: 3; max matches in a query range: 0; matrix: BLOSUM62; gapcosts: existence 11, extension 1; compositional adjustments: conditionalcompositional score matrix adjustment.

Amino acid exchange: The term amino acid exchange refers to the exchangeof a given amino acid residue in an amino acid sequence by any otheramino acid residue having a different chemical structure, preferably byanother proteinogenic amino acid residue. Thus, in contrast to insertionor deletion of an amino acid, the amino acid exchange does not changethe total number of amino acids of said amino acid sequence. Verypreferred in the context of the invention is the exchange of an aminoacid residue of said amino acid sequence to be mutated by a lysineresidue or by a cysteine residue.

Epitope: The term epitope refers to continuous or discontinuous portionsof an antigen, preferably a polypeptide, wherein said portions can bespecifically bound by an antibody or by a T-cell receptor within thecontext of an MHC molecule. With respect to antibodies, specific bindingexcludes non-specific binding but does not necessarily excludecross-reactivity. An epitope typically comprise 5-20 amino acids in aspatial conformation which is unique to the antigenic site.

T helper (Th) cell epitope: The term “T helper (Th) cell epitope” asused herein refers to an epitope that is capable of recognition by ahelper Th cell. In another preferred embodiment, said T helper cellepitope is a universal T helper cell epitope.

Universal Th cell epitope: The term “universal Th cell epitope” as usedherein refers to a Th cell epitope that is capable of binding to atleast one, preferably more than one MHC class II molecules. The simplestway to determine whether a peptide sequence is a universal Th cellepitope is to measure the ability of the peptide to bind to individualMHC class II molecules. This may be measured by the ability of thepeptide to compete with the binding of a known Th cell epitope peptideto the MHC class II molecule. A representative selection of HLA-DRmolecules are described in e.g. Alexander J, et al., Immunity (1994)1:751-761. Affinities of Th cell epitopes for MHC class II moleculesshould be at least 10⁻⁵M. An alternative, more tedious but also morerelevant way to determine the “universality” of a Th cell epitope is thedemonstration that a larger fraction of people (>30%) generate ameasurable T cell response upon immunization and boosting one monthslater with a protein containing the Th cell epitope formulated in IFA. Arepresentative collection of MHC class II molecules present in differentindividuals is given in Panina-Bordignon P, et al., Eur J Immunol (1989)19:2237-2242. As a consequence, the term “universal Th cell epitope” asused herein preferably refers to a Th cell epitope that generates ameasurable T cell response upon immunization and boosting (one monthslater with a protein containing the Th cell epitope formulated in IFA)in more than 30% of a selected group of individuals as described inPanina-Bordignon P, et al., Eur J Immunol (1989) 19:2237-2242. Moreover,and again further preferred, the term “universal Th cell epitope” asused herein preferably refers to a Th cell epitope that is capable ofbinding to at least one, preferably to at least two, and even morepreferably to at least three DR alleles selected from of DR1, DR2w2b,DR3, DR4w4, DR4w14, DR5, DR7, DR52a, DRw53, DR2w2a; and preferablyselected from DR1, DR2w2b, DR4w4, DR4w14, DR5, DR7, DRw53, DR2w2a, withan affinity at least 500 nM (as described in Alexander J, et al.,Immunity (1994) 1:751-761 and references cited herein); a preferredbinding assay to evaluate said affinities is the one described by SetteA, et al., J Immunol (1989) 142:35-40. In an even again more preferablemanner, the term “universal Th cell epitope” as used herein refers to aTh cell epitope that is capable of binding to at least one, preferablyto at least two, and even more preferably to at least three DR allelesselected from DR1, DR2w2b, DR4w4, DR4w14, DR5, DR7, DRw53, DR2w2a, withan affinity at least 500 nM (as described in Alexander J, et al.,Immunity (1994) 1:751-761 and references cited herein); a preferredbinding assay to evaluate said affinities is the one described by SetteA, et al., J Immunol (1989) 142:35-40.

Universal Th cell epitopes are described, and known to the skilledperson in the art, such as by Alexander J, et al., Immunity (1994)1:751-761, Panina-Bordignon P, et al., Eur J Immunol (1989)19:2237-2242, Calvo-Calle J M, et al., J Immunol (1997) 159:1362-1373,and Valmori D, et al., J Immunol (1992) 149:717-721.

Adjuvant: The term “adjuvant” as used herein refers to non-specificstimulators of the immune response or substances that allow generationof a depot in the host which when combined with the vaccine andpharmaceutical composition, respectively, of the present invention mayprovide for an even more enhanced immune response. Preferred adjuvantsare complete and incomplete Freund's adjuvant, aluminum containingadjuvant, preferably aluminum hydroxide, and modified muramyldipeptide.Further preferred adjuvants are mineral gels such as aluminum hydroxide,surface active substances such as lyso lecithin, pluronic polyols,polyanions, peptides, oil emulsions, keyhole limpet hemocyanins,dinitrophenol, and human adjuvants such as BCG (bacille Calmette Guerin)and Corynebacterium parvum. Such adjuvants are also well known in theart. Further adjuvants that can be administered with the compositions ofthe invention include, but are not limited to, Monophosphoryl lipidimmunomodulator, AdjuVax 100a, QS-21, QS-18, CRL1005, Aluminum salts(Alum), MF-59, OM-174, OM-197, OM-294, and Virosomal adjuvanttechnology. The adjuvants may also comprise mixtures of thesesubstances. Virus-like particles have been generally described as anadjuvant. However, the term “adjuvant”, as used within the context ofthis application, refers to an adjuvant not being the inventivevirus-like particle. Rather “adjuvant” relates to an additional,distinct component of the inventive compositions, vaccines orpharmaceutical compositions.

Effective amount: As used herein, the term “effective amount” refers toan amount necessary or sufficient to realize a desired biologic effect.An effective amount of the composition, or alternatively thepharmaceutical composition, would be the amount that achieves thisselected result, and such an amount could be determined as a matter ofroutine by a person skilled in the art. Preferably, the term “effectiveamount”, as used herein, refers to an amount necessary or sufficient tobe effective to reduce the allergenicity of a cat typically andpreferably for a human. Preferably, the term “effective amount”, as usedherein, refers to an amount necessary or sufficient to be effective togenerate immune complexes formed of Fel d1 and Fel d1-antibodies in thesaliva, the fur, the skin or the tears of a cat, preferably in thesaliva of a cat as described herein. The effective amount can varydepending on the particular composition being administered and the sizeof the subject. One of ordinary skill in the art can empiricallydetermine the effective amount of a particular composition of thepresent invention without necessitating undue experimentation.

Treatment: As used herein, the terms “treatment”, “treat”, “treated” or“treating” refer to prophylaxis and/or therapy. In one embodiment, theterms “treatment”, “treat”, “treated” or “treating” refer to atherapeutic treatment. In another embodiment, the terms “treatment”,“treat”, “treated” or “treating” refer to a prophylactic treatment.

Fel d1 protein: The term “Fel d1 protein”, as used herein, refers to aprotein comprising or alternatively consisting of chain 1 of Fel d1 andchain 2 of Fel d1. Preferably chain 1 of Fel d1 and chain 2 of Fel d1are linked covalently. In one preferred embodiment, the chain 1 of Feld1 and chain 2 of Fel d1 are linked via at least one disulfide bond. Inanother preferred embodiment, the chain 1 and chain 2 are fused eitherdirectly or via a spacer, in which case said Fel d1 protein furthercomprises or alternatively consists of a spacer. Preferably the Fel d1protein, as defined herein, consists of at most 300, even morepreferably at most 200 amino acids in total. Typically and preferably,Fel d1 protein, according to the invention, is capable of inducing invivo the production of antibody specifically binding to either thenaturally occurring Fel d1, the endogenous Fel d1 or the recombinant Feld1 fusion proteins as produced according to Example 7-9 of the presentinvention.

Chain 1 of Fel d1: The term “chain 1 of Fel d1”, as used herein, refersto a polypeptide comprising or alternatively consisting of an amino acidsequence as of SEQ ID NO:30 or a homologous sequence thereof. The term“homologous sequence of SEQ ID NO:30”, as used herein, refers to apolypeptide that has an identity to SEQ ID NO:30 which is greater than80%, more preferably greater than 90%, and even more preferably greaterthan 95%. The term “chain 1 of Fel d1”, as used herein, should alsorefer to a polypeptide encompassing at least one post-translationalmodification, including but not limited to at least one glycosylation,of chain 1 of Fel d1, as defined herein. Preferably the chain 1 of Feld1, as defined herein, consists of at most 130, even more preferably atmost 100 amino acids in total.

Chain 2 of Fel d1: The term “chain 2 of Fel d1”, as used herein, refersto a polypeptide comprising or alternatively consisting of an amino acidsequence as of SEQ ID NO:31, SEQ ID NO:32 or SEQ ID NO:33, or ahomologous sequence thereof. The term “homologous sequence of SEQ IDNO:31, SEQ ID NO:32 or SEQ ID NO:33, as used herein, refers to apolypeptide that has an identity to SEQ ID NO:31, SEQ ID NO:32 or SEQ IDNO:33 which is greater than 80%, more preferably greater than 90%, andeven more preferably greater than 95%. The term “chain 2 of Fel d1”, asused herein, should also refer to a polypeptide encompassing at leastone post-translational modification, including but not limited to atleast one glycosylation, of chain 2 of Fel d1, as defined hereinPreferably the chain 2 of Fel d1, as defined herein, consists of at most150, even more preferably at most 130, still more preferably at most 100amino acids in total.

Immune complex: The term “immune complex”, as used herein, refers to acomplex formed from the binding of antibody to its cognate/specificantigen. Preferably, the term “immune complex”, as used herein, refersto a complex formed from the non-covalent binding of antibody to itscognate/specific antigen. Further preferably, the term “immune complex”,as used herein, refers to a complex formed from the binding, preferablythe non-covalent binding, of Fel d1-antibody to Fel d1.

Attachment Site, First: As used herein, the phrase “first attachmentsite” refers to an element which is naturally occurring with thevirus-like particle or which is artificially added to the virus-likeparticle, and to which the second attachment site may be linked. Thefirst attachment site preferably is a protein, a polypeptide, an aminoacid, a peptide, a sugar, a polynucleotide, a natural or syntheticpolymer, a secondary metabolite or compound (biotin, fluorescein,retinol, digoxigenin, metal ions, phenylmethylsulfonylfluoride), or achemically reactive group such as an amino group, a carboxyl group, asulfhydryl group, a hydroxyl group, a guanidinyl group, histidinylgroup, or a combination thereof. A preferred embodiment of a chemicallyreactive group being the first attachment site is the amino group of anamino acid residue, preferably of a lysine residue. The first attachmentsite is typically located on the surface, and preferably on the outersurface of the VLP. Multiple first attachment sites are present on thesurface, preferably on the outer surface of the VLP, typically in arepetitive configuration. In a preferred embodiment the first attachmentsite is associated with the VLP, through at least one covalent bond,preferably through at least one peptide bond. In a further preferredembodiment the first attachment site is naturally occurring with theVLP. Alternatively, in a preferred embodiment the first attachment siteis artificially added to the VLP. In a very preferred embodiment saidfirst attachment site is the amino group of a lysine residue of theamino acid sequence of said VLP polypeptide.

Attachment Site, Second: As used herein, the phrase “second attachmentsite” refers to an element which is naturally occurring with or which isartificially added to the Fel d1 protein, and to which the firstattachment site may be linked. The second attachment site of the Fel d1protein preferably is a protein, a polypeptide, a peptide, an aminoacid, a sugar, a polynucleotide, a natural or synthetic polymer, asecondary metabolite or compound (biotin, fluorescein, retinol,digoxigenin, metal ions, phenylmethylsulfonylfluoride), or a chemicallyreactive group such as an amino group, a carboxyl group, a sulfhydrylgroup, a hydroxyl group, a guanidinyl group, histidinyl group, or acombination thereof. A preferred embodiment of a chemically reactivegroup being the second attachment site is a sulfhydryl group, preferablythe sulfhydryl group of the amino acid cysteine most preferably thesulfhydryl group of a cysteine residue. The term “antigen with at leastone second attachment site” or “Fel d1 protein with at least one secondattachment site” refers, therefore, to a construct comprising the Fel d1protein and at least one second attachment site. However, in particularfor a second attachment site, which is not naturally occurring withinthe Fel d1 protein, such a construct typically and preferably furthercomprises a “linker”. In another preferred embodiment the secondattachment site is associated with the Fel d1 protein through at leastone covalent bond, preferably through at least one peptide bond. In afurther embodiment, the second attachment site is naturally occurringwithin the Fel d1 protein. In another further preferred embodiment, thesecond attachment site is artificially added to the Fel d1 proteinthrough a linker, wherein said linker comprises or alternativelyconsists of a cysteine. Preferably, the linker is fused to the Fel d1protein by a peptide bond.

Linked: The terms “linked” or “linkage” as used herein, refer to allpossible ways, preferably chemical interactions, by which the at leastone first attachment site and the at least one second attachment siteare joined together. Chemical interactions include covalent andnon-covalent interactions. Typical examples for non-covalentinteractions are ionic interactions, hydrophobic interactions orhydrogen bonds, whereas covalent interactions are based, by way ofexample, on covalent bonds such as ester, ether, phosphoester,carbon-phosphorus bonds, carbon-sulfur bonds such as thioether, or imidebonds. In certain preferred embodiments the first attachment site andthe second attachment site are linked through at least one covalentbond, preferably through at least one non-peptide bond, and even morepreferably through exclusively non-peptide bond(s). The term “linked” asused herein, however, shall not only refer to a direct linkage of the atleast one first attachment site and the at least one second attachmentsite but also, alternatively and preferably, an indirect linkage of theat least one first attachment site and the at least one secondattachment site through intermediate molecule(s), and hereby typicallyand preferably by using at least one, preferably one, heterobifunctionalcross-linker. In other preferred embodiments the first attachment siteand the second attachment site are linked through at least one covalentbond, preferably through at least one peptide bond, and even morepreferably through exclusively peptide bond(s).

Linker: A “linker”, as used herein, either associates the secondattachment site with the Fel d1 protein or already comprises,essentially consists of, or consists of the second attachment site.Preferably, a “linker”, as used herein, already comprises the secondattachment site, typically and preferably—but not necessarily—as oneamino acid residue, preferably as a cysteine residue. A preferredlinkers are an amino acid linkers, i.e. linkers containing at least oneamino acid residue. The term amino acid linker does not imply that sucha linker consists exclusively of amino acid residues. However, a linkerconsisting exclusively of amino acid residues is a preferred embodimentof the invention. The amino acid residues of the linker are, preferably,composed of naturally occurring amino acids or unnatural amino acidsknown in the art, all-L or all-D or mixtures thereof. Further preferredembodiments of a linker in accordance with this invention are moleculescomprising a sulfhydryl group or a cysteine residue and such moleculesare, therefore, also encompassed within this invention. Association ofthe linker with the Fel d1 protein is preferably by way of at least onecovalent bond, more preferably by way of at least one peptide bond.

Thus, in a first aspect, the present invention provides for an use of acomposition in a method of reducing the allergenicity of a cat, whereinan effective amount of said composition is administered to said cat, andwherein said composition comprises (i) a virus-like particle with atleast one first attachment site; (ii) at least one Fel d1 protein withat least one second attachment site; and wherein said virus-likeparticle and said Fel d1 protein are linked through said at least onefirst and said at least one second attachment site. Preferably, saidmethod is a non-therapeutic method of reducing the allergenicity of saidcat. In a further preferred embodiment, said cat is not suffering froman allergy or an auto-immune disease, preferably wherein said cat is notsuffering from an allergy or an auto-immune disease caused by Fel d1.

In a preferred embodiment, said reducing the allergenicity of said cat,typically and preferably for a human, is effected by generating immunecomplexes formed of Fel d1 and Fel d1-antibodies in the saliva, the fur,the skin or the tears of said cat, preferably in the saliva of said cat,and wherein preferably said administration of said composition leads tosaid generating of said immune complexes in the saliva, fur, skin ortears of said cat, preferably in the saliva of said cat.

The reduction of the allergenicity of said cat for a human caused by theadministration of the inventive compositions to said cat can further bedetermined by way of degranulation of basophils from cat allergicpatients as described in the examples. Thus, In a preferred embodiment,said reducing the allergenicity of said cat for a human, is reducing theallergenicity of the Fel d1 shed by said cat, and wherein preferablysaid reducing the allergenicity of the Fel d1 shed by said cat isreducing the allergenicity of the Fel d1 in the saliva, the fur, theskin or the tears of said cat, preferably in the saliva of said cat.

In a preferred embodiment, said administering of said effective amountof the composition to the cat comprises repeated administrations of saideffective amount of the composition to the cat, and wherein saidrepeated administrations are effected in intervals of 2, 3, 4, 8, 12weeks, and wherein preferably said repeated administrations comprise 2,3, 4 or 5 administrations of said effective amount of the composition tothe cat.

In a further preferred embodiment, said repeated administrations arethree administrations effected in intervals of 3 or 4 weeks. Typicallyand preferably said administering of said effective amount of thecomposition to the cat further comprises a single administration of saideffective amount of the composition to the cat, wherein said singleadministration is effected 6, 9, 12, 15 or 18 months, preferably 12months, after the last of said repeated administrations.

Typically, said reduction of said allergically active Fel d1 in thesaliva, fur, skin or tears of said cat, preferably in the saliva of saidcat, is present at least between one month and 3 months after the lastof said repeated administrations.

In a further very preferred embodiment, said reducing the allergenicityof said cat is reducing the allergenicity of said cat for a humanexposed to said cat. In a further very preferred embodiment, saidreducing the allergenicity of said cat for said human exposed to the catis (i) reducing the level or severity of the allergic response generatedby said human, or (ii) reducing at least one allergic symptom of saidhuman; and wherein preferably said exposure of said human to said cat isthe exposure of said human to the saliva, fur, skin or tears of saidcat, preferably to the saliva of said cat.

In a further very preferred embodiment, said reducing the allergenicityof said cat is reducing the allergenicity of said cat for a humanexposed to said cat, wherein said reducing the allergenicity of said catfor said human exposed to the cat is (i) reducing the level or severityof the allergic response generated by said human, or (ii) reducing atleast one allergic symptom of said human; and wherein preferably saidexposure of said human to said cat is the exposure of said human to thesaliva, fur, skin or tears of said cat, preferably to the saliva of saidcat. Preferably, (i) said reduction in the level or severity of theallergic response generated by said human, or (ii) said reduction ofsaid at least one allergic symptom of said human, is expressed by a lesspositive symptom score test, skin prick test, nasal provocation test orconjunctival provocation test, preferably by a less positive symptomscore test or skin prick test, wherein preferably the saliva, fur, skinor tears from said cat before and after said administration, furtherpreferably the saliva from said cat before and after saidadministration, is used for said skin prick test, nasal provocation testor conjunctival provocation test, preferably said symptom score test orsaid skin prick test. It is known to the skilled person in the art thatallergy and allergic symptoms can be assessed using a symptom scoretest, skin prick test, a nasal provocation test, a conjunctivalprovocation test or a bronchial provocation test. These procedures,questionnaires and tests are well-known to the skilled in the art. Theterm “less positive” as used herein and in the context of a symptomscore test, skin prick test, a nasal provocation test, a conjunctivalprovocation test, and in particular in the context of a symptom scoretest or a skin prick test refers to a (i) lower or reduced level orseverity of the allergic response generated by said human upon exposureto the saliva, fur, skin or tears of said cat, preferably to the salivaof said cat or (ii) lowering or reduction of at least one allergicsymptom of said human upon exposure to said cat, preferably uponexposure to the saliva, fur, skin or tears of said cat, preferably tothe saliva of said cat, and more preferably upon exposure to the salivaof said cat.

In one embodiment, said virus-like particle is derived from a virusbeing non-pathogenic to said cat. In a preferred embodiment, saidvirus-like particle (VLP) is derived from a plant virus or abacteriophage, and wherein preferably said bacteriophage is derived froma RNA bacteriophage, and wherein further preferably said VLP is derivedfrom a RNA bacteriophage or a plant virus, and again further preferablywherein said VLP is derived from a plant virus. In another preferredembodiment, said VLP is a recombinant VLP, and wherein preferably saidrecombinant VLP is derived from a plant virus. In another preferredembodiment, said VLP is a VLP of cucumber mosaic virus (CMV). In anotherpreferred embodiment, said VLP is a VLP of an RNA bacteriophage,preferably said VLP is a recombinant VLP of an RNA bacteriophage. Inanother preferred embodiment, said virus-like particle is a virus-likeparticle of an RNA-bacteriophage Q. In another preferred embodiment,said VLP is not a VLP of an RNA bacteriophage, preferably said VLP isnot a recombinant VLP of an RNA bacteriophage. In another preferredembodiment, said virus-like particle is not a virus-like particle of anRNA-bacteriophage Qβ.

In a preferred embodiment, said VLP is a modified VLP comprising,essentially consisting of, or alternatively consisting of, at least onemodified VLP polypeptide, wherein said modified VLP polypeptidecomprises, or preferably consists of, (a) a VLP polypeptide, and (b) a Thelper cell epitope, wherein said VLP polypeptide comprises, orpreferably consists of, (i) an amino acid sequence of a coat protein ofa virus, preferably an amino acid sequence of a coat protein of a plantvirus; or (ii) a mutated amino acid sequence, wherein the amino acidsequence to be mutated is an amino acid sequence of said coat protein ofa virus, and wherein said mutated amino acid sequence and said coatprotein of a virus show a sequence identity of at least 90%, preferablyof at least 95%, further preferably of at least 98% and again morepreferably of at least 99%.

In a preferred embodiment, said VLP is a modified VLP of cucumber mosaicvirus (CMV), wherein said modified VLP of CMV comprises, essentiallyconsists of, or alternatively consists of, at least one modified CMVpolypeptide, wherein said modified CMV polypeptide comprises, orpreferably consists of, (a) a CMV polypeptide, and (b) a T helper cellepitope; and wherein said CMV polypeptide comprises, or preferablyconsists of, (i) an amino acid sequence of a coat protein of CMV; or(ii) a mutated amino acid sequence, wherein the amino acid sequence tobe mutated is an amino acid sequence of a coat protein of CMV, andwherein said mutated amino acid sequence and said coat protein of CMVshow a sequence identity of at least 90%, preferably of at least 95%,further preferably of at least 98% and again more preferably of at least99%.

In a preferred embodiment, said CMV polypeptide comprises, preferablyconsists of, an amino acid sequence of a coat protein of CMV. In anotherpreferred embodiment, said CMV polypeptide comprises, preferablyconsists of a mutated amino acid sequence, wherein the amino acidsequence to be mutated is an amino acid sequence of a coat protein ofCMV, and wherein said mutated amino acid sequence and said coat proteinof CMV show a sequence identity of at least 90%, preferably of at least95%, further preferably of at least 98% and again more preferably of atleast 99%. Typically and preferably, said mutated amino acid sequenceand said amino acid sequence to be mutated differ in least one and in atmost 11, 10, 9, 8, 7, 6, 5, 4, 3, or 2 amino acid residues, and whereinpreferably these differences are selected from (i) insertion, (ii)deletion, (iii) amino acid exchange, and (iv) any combination of (i) to(iii).

In another preferred embodiment, said CMV polypeptide comprises, orpreferably consists of, (i) (a) an amino acid sequence of a coat proteinof CMV, wherein said amino acid sequence comprises, or preferablyconsists of, SEQ ID NO:1 or (b) an amino acid sequence having a sequenceidentity of at least 75%, preferably of at least 80%, more preferably ofat least 85%, again further preferably of at least 90%, again morepreferably of at least 95%, still further preferably of at least 98% andstill again further more preferably of at least 99% of SEQ ID NO:1; or(ii) a mutated amino acid sequence, wherein said amino acid sequence tobe mutated is said amino acid sequence as defined in (i) of this claim,and wherein said mutated amino acid sequence and said amino acidsequence to be mutated show a sequence identity of at least 95%,preferably of at least 98%, and more preferably of at least 99%.

In another preferred embodiment, said CMV polypeptide comprises, orpreferably consists of, (a) an amino acid sequence of a coat protein ofCMV, wherein said amino acid sequence comprises, or preferably consistsof, SEQ ID NO:1 or (b) an amino acid sequence having a sequence identityof at least 75%, preferably of at least 80%, more preferably of at least85%, again further preferably of at least 90%, again more preferably ofat least 95%, still further preferably of at least 98% and still againfurther more preferably of at least 99% of SEQ ID NO:1.

In another preferred embodiment, said CMV polypeptide comprises, orpreferably consists of, (i) (a) an amino acid sequence of a coat proteinof CMV, wherein said amino acid sequence comprises SEQ ID NO:34, or (b)an amino acid sequence of a coat protein of CMV comprising an amino acidsequence region, wherein said amino acid sequence region has a sequenceidentity of at least 75%, preferably of at least 80%, more preferably ofat least 85%, again further preferably of at least 90%, again morepreferably of at least 95%, still further preferably of at least 98% andstill again further more preferably of at least 99% with SEQ ID NO:34;or (ii) a mutated amino acid sequence, wherein said amino acid sequenceto be mutated is said amino acid sequence as defined in (i) of thisclaim, and wherein said mutated amino acid sequence and said amino acidsequence to be mutated show a sequence identity of at least 95%,preferably of at least 98%, and more preferably of at least 99%.

In a further preferred embodiment, said CMV polypeptide comprises, orpreferably consists of, (a) an amino acid sequence of a coat protein ofCMV, wherein said amino acid sequence comprises SEQ ID NO:34, or (b) anamino acid sequence of a coat protein of CMV comprising an amino acidsequence region, wherein said amino acid sequence region has a sequenceidentity of at least 75%, preferably of at least 80%, more preferably ofat least 85%, again further preferably of at least 90%, again morepreferably of at least 95%, still further preferably of at least 98% andstill again further more preferably of at least 99% with SEQ ID NO:34.

In another preferred embodiment, said CMV polypeptide comprises, orpreferably consists of, (i) (a) an amino acid sequence of a coat proteinof CMV, wherein said amino acid sequence comprises, or preferablyconsists of, SEQ ID NO:1 or (b) an amino acid sequence having a sequenceidentity of at least 75%, preferably of at least 80%, more preferably ofat least 85%, again further preferably of at least 90%, again morepreferably of at least 95%, still further preferably of at least 98% andstill again further more preferably of at least 99% of SEQ ID NO:1; andwherein said amino sequence as defined in (a) or (b) in this claimcomprises SEQ ID NO:34; or wherein said amino sequence as defined in (a)or (b) in this claim comprises an amino acid sequence region, whereinsaid amino acid sequence region has a sequence identity of at least 75%,preferably of at least 80%, more preferably of at least 85%, againfurther preferably of at least 90%, again more preferably of at least95%, still further preferably of at least 98% and still again furthermore preferably of at least 99% with SEQ ID NO:34; or (ii) a mutatedamino acid sequence, wherein said amino acid sequence to be mutated issaid amino acid sequence as defined in (i) of this claim, and whereinsaid mutated amino acid sequence and said amino acid sequence to bemutated show a sequence identity of at least 98% preferably of at least99%.

In another preferred embodiment, said CMV polypeptide comprises, orpreferably consists of, (a) an amino acid sequence of a coat protein ofCMV, wherein said amino acid sequence comprises, or preferably consistsof, SEQ ID NO:1 or (b) an amino acid sequence having a sequence identityof at least 90% of SEQ ID NO:1; and wherein said amino sequence asdefined in (a) or (b) in this claim comprises SEQ ID NO:34; or whereinsaid amino sequence as defined in (a) or (b) in this claim comprises anamino acid sequence region, wherein said amino acid sequence region hasa sequence identity of at least 90% with SEQ ID NO:34.

In another preferred embodiment, said T helper cell epitope replaces aN-terminal region of said CMV polypeptide. In another preferredembodiment the number of amino acids of said N-terminal region replacedis equal to or lower than the number of amino acids of which said Thelper cell epitope consists.

In a further very preferred embodiment, said T helper cell epitopereplaces a N-terminal region of said CMV polypeptide, and wherein thenumber of amino acids of said N-terminal region replaced is equal to orlower than the number of amino acids of which said T helper cell epitopeconsists. Typically and preferably, said replaced N-terminal region ofsaid CMV polypeptide consists of 5 to 15 consecutive amino acids,preferably of 9 to 14 consecutive amino acids, more preferably of 11 to13 consecutive amino acids.

In a further very preferred embodiment, said N-terminal region of saidCMV polypeptide corresponds to amino acids 2-12 of SEQ ID NO: 1.

In another very preferred embodiment, said T helper cell epitope is auniversal T helper cell epitope. In another preferred embodiment, said Thelper cell epitope consists of at most 20 amino acids.

In a very preferred embodiment, said Th cell epitope is a PADREsequence. In a further very referred embodiment, said Th cell epitopecomprises, preferably consists of, the amino acid sequence of SEQ IDNO:5. In another very preferred embodiment, said Th cell epitope is aPADRE sequence, and wherein said Th cell epitope comprises, preferablyconsists of, the amino acid sequence of SEQ ID NO:5.

In another preferred embodiment, said T helper cell epitope is derivedfrom a human vaccine. In a very preferred embodiment, said Th cellepitope is derived from tetanus toxin. In a further very referredembodiment, said Th cell epitope has, preferably consists of, the aminoacid sequence of SEQ ID NO:4. In another very preferred embodiment, saidTh cell epitope is derived from tetanus toxin, and wherein said Th cellepitope has, preferably consists of, the amino acid sequence of SEQ IDNO:4.

In a very preferred embodiment, said Th cell epitope is a PADREsequence, and wherein said Th cell epitope comprises, preferablyconsists of, the amino acid sequence of SEQ ID NO:5; or wherein said Thcell epitope is derived from tetanus toxin, and wherein said Th cellepitope has, preferably consists of, the amino acid sequence of SEQ IDNO:4.

In a very preferred embodiment, said CMV polypeptide comprises, orpreferably consists of, an amino acid sequence of a coat protein of CMV,wherein said amino acid sequence comprises, or preferably consists of,SEQ ID NO:1 or an amino acid sequence having a sequence identity of atleast 95% of SEQ ID NO:1; and wherein said amino sequence comprises SEQID NO:34, and wherein said T helper cell epitope replaces the N-terminalregion of said CMV polypeptide, and wherein said replaced N-terminalregion of said CMV polypeptide consists of 11 to 13 consecutive aminoacids, preferably of 11 consecutive amino acids, and wherein furtherpreferably said N-terminal region of said CMV polypeptide corresponds toamino acids 2-12 of SEQ ID NO: 1.

In another very preferred embodiment, said modified CMV polypeptidecomprises, preferably consists of, an amino acid sequence of SEQ IDNO:6. In another very preferred embodiment, said modified CMVpolypeptide comprises, preferably consists of, an amino acid sequence ofSEQ ID NO:7. The use of a composition of any one of the claims 6 to 8,wherein said modified CMV polypeptide comprises, preferably consists of,an amino acid sequence of SEQ ID NO:6 or SEQ ID NO:7.

In a very preferred embodiment, said first attachment site and saidsecond attachment site are linked via at least one covalentnon-peptide-bond. In another very preferred embodiment, said firstattachment site comprises, or preferably is, an amino group, preferablyan amino group of a lysine. In a further very preferred embodiment, saidsecond attachment site comprises, or preferably is, a sulfhydryl group,preferably a sulfhydryl group of a cysteine.

In a very preferred embodiment, the at least one first attachment siteis an amino group, preferably an amino group of a lysine residue and theat least one second attachment site is a sulfhydryl group, preferably asulfhydryl group of a cysteine residue or a sufhydryl group that hasbeen chemically attached to the Fel d1 protein. In a further preferredembodiment only one of said second attachment sites associates with saidfirst attachment site through at least one non-peptide covalent bondleading to a single and uniform type of binding of said Fel d1 proteinto said modified virus-like particle, wherein said only one secondattachment site that associates with said first attachment site is asulfhydryl group, and wherein said Fel d1 protein and said modifiedvirus-like particle interact through said association to form an orderedand repetitive antigen array, i.e. an ordered and repetitive array ofFel d1 proteins.

In one preferred embodiment of the invention, the Fel d1 protein islinked to the modified VLP by way of chemical cross-linking, typicallyand preferably by using a heterobifunctional cross-linker. In preferredembodiments, the hetero-bifunctional cross-linker contains a functionalgroup which can react with the preferred first attachment sites,preferably with the amino group, more preferably with the amino groupsof lysine residue(s) of the modified VLP, and a further functional groupwhich can react with the preferred second attachment site, i.e. asulfhydryl group, preferably of cysteine(s) residue inherent of, orartificially added to the Fel d1 protein, and optionally also madeavailable for reaction by reduction. Several hetero-bifunctionalcross-linkers are known to the art. These include the preferredcross-linkers SMPH (Pierce), Sulfo-MBS, Sulfo-EMCS, Sulfo-GMBS,Sulfo-SIAB, Sulfo-SMPB, Sulfo-SMCC, Sulfo-KMUS SVSB, SIA, and othercross-linkers available for example from the Pierce Chemical Company,and having one functional group reactive towards amino groups and onefunctional group reactive towards sulfhydryl groups. The above mentionedcross-linkers all lead to formation of an amide bond after reaction withthe amino group and a thioether linkage with the sulfhydryl groups.Another class of cross-linkers suitable in the practice of the inventionis characterized by the introduction of a disulfide linkage between theFel d1 protein and the modified VLP upon coupling. Preferredcross-linkers belonging to this class include, for example, SPDP andSulfo-LC-SPDP (Pierce).

Linking of the Fel d1 protein to the modified VLP by using ahetero-bifunctional cross-linker according to the preferred methodsdescribed above, allows coupling of the Fel d1 protein to the modifiedVLP in an oriented fashion. Other methods of linking the Fel d1 proteinto the modified VLP include methods wherein the Fel d1 protein iscross-linked to the modified VLP, using the carbodiimide EDC, and NHS.The Fel d1 protein may also be first thiolated through reaction, forexample with SATA, SATP or iminothiolane. The Fel d1 protein, afterdeprotection if required, may then be coupled to the modified VLP asfollows. After separation of the excess thiolation reagent, the Fel d1protein is reacted with the modified VLP, previously activated with ahetero-bifunctional cross-linker comprising a cysteine reactive moiety,and therefore displaying at least one or several functional groupsreactive towards cysteine residues, to which the thiolated Fel d1protein can react, such as described above. Optionally, low amounts of areducing agent are included in the reaction mixture. In further methods,the Fel d1 protein is attached to the modified VLP, using ahomo-bifunctional cross-linker such as glutaraldehyde, DSG, BM[PEO]4,BS3, (Pierce) or other known homo-bifunctional cross-linkers withfunctional groups reactive towards amine groups or carboxyl groups ofthe modified VLP.

In very preferred embodiments of the invention, the Fel d1 protein islinked via a cysteine residue, having been added to either theN-terminus or the C-terminus of, or a natural cysteine residue withinthe Fel d1 protein, to lysine residues of the modified virus-likeparticle. In a preferred embodiment, the composition of the inventionfurther comprises a linker, wherein said linker associates said Fel d1protein with said second attachment site, and wherein preferably saidlinker comprises or alternatively consists of said second attachmentsite.

In another very preferred embodiment, said composition further comprisesa linker, said linker is fused to the C-terminus of said Fel d1 protein.In a very preferred embodiment, said Fel d1 protein comprises chain 1 ofFel d1 and chain 2 of Fel d1, wherein said chain 1 of Fel d1 isassociated with chain 2 of Fel d1 by at least one covalent bond.

In a very preferred embodiment, said Fel d1 protein is a Fel d1 fusionprotein comprising chain 1 of Fel d1 and chain 2 of Fel d1, whereinchain 1 of Fel d1 and chain 2 of Fel d1 are fused either directly viaone peptide bond or via a spacer, which links the N-terminus of onechain with the C-terminus of another chain. Several recombinant fusionproteins of Fel d1 have been described (Vailes L D, et al., J AllergyClin Immunol (2002) 110:757-762; Gronlund H, et al., J Biol Chem (2003)278:40144-40151; Schmitz N, et al., J Exp Med (2009) 206:1941-1955;WO2006/097530). In a further preferred embodiment, said Fel d1 proteinis a Fel d1 fusion protein comprising chain 1 of Fel d1 and chain 2 ofFel d1, wherein said chain 2 of Fel d1 is fused via its C-terminus tothe N-terminus of said chain 1 of Fel d1 either directly via one peptidebond or via a spacer, wherein said spacer consists of an amino acidsequence having 1-20 amino acid residues, wherein preferably said spacerconsists of an amino acid sequence having 10-20 amino acid residues. Inanother very preferred embodiment, said spacer consists of an amino acidsequence of 15 amino acid residues, and wherein preferably said spacerhas an amino acid sequence of SEQ ID NO: 17.

In a further very preferred embodiment, said Fel d1 protein is a Fel d1fusion protein comprising chain 1 of Fel d1 and chain 2 of Fel d1,wherein said chain 1 of Fel d1 is fused via its C-terminus to theN-terminus of said chain 2 of Fel d1 either directly via one peptidebond or via a spacer, wherein said spacer consists of an amino acidsequence having 1-20 amino acid residues, wherein preferably said spacerconsists of an amino acid sequence having 10-20 amino acid residues. Inanother very preferred embodiment, said spacer consists of an amino acidsequence of 15 amino acid residues, and wherein preferably said spacerhas an amino acid sequence of SEQ ID NO: 17.

In another very preferred embodiment, said chain 1 of Fel d 1 comprisesa sequence of SEQ ID NO:30 or a homologue sequence thereof, wherein saidhomologue sequence has an identity to SEQ ID NO:30 of greater than 80%,preferably greater than 90%, or even more preferably greater than 95%.Preferably, said chain 1 of Fel d 1 comprises a sequence of SEQ ID NO:30or a homologue sequence thereof, wherein said homologue sequence has anidentity to SEQ ID NO:30 of greater than 90%, or even more preferablygreater than 95%.

In another very preferred embodiment, said chain 2 of Fel d 1 comprisesa sequence of SEQ ID NO:31, SEQ ID NO:32 or SEQ ID NO:33, or a homologuesequence thereof, wherein said homologue sequence has an identity to SEQID NO:31, SEQ ID NO:32 or SEQ ID NO:33 of greater than 80%, preferablygreater than 90%, and even more preferably greater than 95%. Furtherpreferably, said chain 2 of Fel d 1 comprises a sequence of SEQ IDNO:31, SEQ ID NO:32 or SEQ ID NO:33, or a homologue sequence thereof,wherein said homologue sequence has an identity to SEQ ID NO:31, SEQ IDNO:32 or SEQ ID NO:33 of greater than 90%, and even more preferablygreater than 95%.

In a very preferred embodiment, said Fel d1 protein comprises an aminoacid sequence selected from: (a) SEQ ID NO:20; (b) SEQ ID NO:25; (c) SEQID NO:26; (d) SEQ ID NO:27; or (e) SEQ ID NO:29. In another verypreferred embodiment, said Fel d1 protein comprises, preferably consistsof, an amino acid sequence of SEQ ID NO:29. In another very preferredembodiment, said Fel d1 protein comprises, preferably consists of, anamino acid sequence of SEQ ID NO:20. In another very preferredembodiment, said Fel d1 protein comprises, preferably consists of, anamino acid sequence of SEQ ID NO:25. In another very preferredembodiment, said Fel d1 protein comprises, preferably consists of, anamino acid sequence of SEQ ID NO:26. In another very preferredembodiment, said Fel d1 protein comprises, preferably consists of, anamino acid sequence of SEQ ID NO:27.

In another aspect, the present invention provides for a method forreducing the allergenicity of a cat, wherein said method comprisesadministering an effective amount of said composition to said cat,wherein said composition comprises (i) a virus-like particle with atleast one first attachment site; (ii) at least one Fel d1 protein withat least one second attachment site; and wherein said virus-likeparticle and said Fel d1 protein are linked through said at least onefirst and said at least one second attachment site. Preferably, saidmethod is a non-therapeutic method of reducing the allergenicity of saidcat; wherein preferably said method or said composition is furtherdefined as described herein.

In a further aspect, the present invention provides for a compositioncomprising (i) a virus-like particle (VLP) with at least one firstattachment site; (ii) at least one Fel d1 protein with at least onesecond attachment site; and wherein said virus-like particle and saidFel d1 protein are linked through said at least one first and said atleast one second attachment site, and wherein said Fel d1 proteincomprises an amino acid sequence selected from SEQ ID NO:25 or SEQ IDNO:27; and wherein said VLP is a modified VLP of cucumber mosaic virus(CMV), wherein said modified VLP of CMV comprises, essentially consistsof, or alternatively consists of, at least one modified CMV polypeptide,wherein said modified CMV polypeptide comprises, or preferably consistsof, (a) a CMV polypeptide, and (b) a T helper cell epitope; and whereinsaid modified CMV polypeptide comprises, preferably consists of, anamino acid sequence of SEQ ID NO:6 or SEQ ID NO:7.

EXAMPLES Example 1 Isolation and Cloning of a Coat Protein (CP) ofCucumber Mosaic Virus (CMV)

Total RNA from CMV-infected lily leaves was isolated using TRI reagent(Sigma, Saint Louis, USA) in accordance with manufacturer'sinstructions. For cDNA synthesis, a OneStep RT-PCR kit (Qiagen, Venlo,Netherlands) was used. For amplification of the CMV CP gene, primersequences were chosen following analysis of CMV sequences from GenBank:CMcpF (CACCATGGACAAATCTGAATCAACCAGTGCTGGT) (SEQ ID NO:8) and CMcpR(CAAAGCTTATCAAACTGGGAGCACCCCAGATGTGGGA) (SEQ ID NO:9); NcoI and HindIIIsites are underlined. The corresponding PCR products were cloned intothe pTZ57R/T vector (Fermentas, Vilnius, Lithuania). E. coli XL1-Bluecells were used as a host for cloning and plasmid amplification. Toavoid selecting clones containing PCR errors, several CP gene-containingpTZ57 plasmid clones were sequenced using a BigDye cycle sequencing kitand an ABI Prism 3100 Genetic analyzer (Applied Biosystems, Carlsbad,USA). After sequencing, a cDNA of the CMV CP gene without sequenceerrors (SEQ ID NO:10) coding for CMV coat protein of SEQ ID NO:1 wasthen subcloned into the NcoI/HindIII sites of the pET28a(+) expressionvector (Novagen, San Diego, USA), resulting in the expression plasmidpET-CMVwt (FIG. 1).

Example 2 Expression of CP of SEQ ID NO:1 in E. coli Leading to VLPs ofCMV

To obtain CMV VLPs, E. coli C2566 cells (New England Biolabs, Ipswich,USA) were transformed with the CMV CP gene-containing plasmid pET-CMVwt.After selection of clones with the highest expression levels of targetprotein, E. coli cultures were grown in 2×TY medium containing kanamycin(25 mg/1) on a rotary shaker (200 rev/min; Infors, Bottmingen,Switzerland) at 30° C. to an OD600 of 0.8-1.0. Then, the cells wereinduced with 0.2 mM IPTG, and the medium was supplemented with 5 mMMgCl2. Incubation was continued on the rotary shaker at 20° C. for 18 h.The resulting biomass was collected by low-speed centrifugation and wasfrozen at −20° C. After thawing on ice, the cells were suspended in thebuffer containing 50 mM sodium citrate, 5 mM sodium borate, 5 mM EDTA, 5mM mercaptoethanol (pH 9.0, buffer A) and were disrupted by ultrasonictreatment. Insoluble proteins and cell debris were removed bycentrifugation (13,000 rpm, 30 min at 5° C.). The soluble CMV CP proteinin clarified lysate was pelleted using saturated ammonium sulfate (1:1,vol/vol) overnight at +4° C. Precipitated proteins were solubilized inthe same buffer A (without mercaptoethanol) for 4 h at +4° C. Insolubleproteins were removed by low speed centrifugation (13,000 rpm, 15 min at4° C.). Soluble CMV CP-containing protein solution was separated fromthe cellular proteins by ultracentrifugation (SW28 rotor, Beckman, PaloAlto, USA; at 25,000 rpm, 6 h, 5° C.) in a sucrose gradient (20-60%sucrose in buffer A, without mercaptoethanol, supplemented with 0.5%Triton X-100). The gradient was divided into six fractions, starting atthe bottom of the gradient, and the fractions were analyzed by SDS-PAGE(data not shown). Fractions No. 2 and No. 3 containing recombinant CMVCP were combined and were dialyzed against 200 volumes of the buffer (5mM sodium borate, 2 mM EDTA, pH 9.0) to remove the sucrose and TritonX-100. After dialysis, CMV CP solution was sterilized by filtrationthrough the 0.2μ filter. Next, CMV CP was concentrated using Type70rotor (Beckman, Palo Alto, USA) ultracentrifugation through the 20%sucrose “cushion” under sterile conditions (50 000 rpm, 4 h, +5° C.).The concentration of purified CMVwt was estimated using the QuBitfluorometer in accordance with manufacturer's recommendations(Invitrogen, Eugene, USA). Concentrated VLP solutions (approx. 3 mg/ml)were stored at +4° C. in 5 mM sodium borate, 2 mM EDTA, buffer (pH 9.0).All steps involved in the expression and purification of VLP weremonitored by SDS-PAGE using 12.5% gels.

CMV coat protein can be successfully expressed in E. coli cells andsignificant part obtained can be in soluble fraction. Moreover, theseproteins are found directly in E. coli cell extracts in the form ofisometric VLPs, as demonstrated by sucrose gradient analysis (FIG. 2A),dynamic light scattering and electron-microscopy analysis (FIG. 2B).

Example 3 Cloning of a Modified Coat Protein of CMV Containing anTetanus Toxoid Epitope (CMV-Ntt830)

To replace the original amino acids at the N-terminus of CMV CP of SEQID NO:1 with the tetanus toxoid epitope coding sequence, the pET-CMVwtplasmid was used for PCR amplification and mutagenesis. A SalI sitelocated within the CMVwt gene (FIG. 1) was used for cloning thecorresponding PCR products.

To introduce the tetanus toxoid epitope coding sequence into the CMVwtgene, a two step PCR mutagenesis was used. For the first stepamplification, the following primers were used: pET-220(AGCACCGCCGCCGCAAGGAA (SEQ ID NO: 11)-upstream from polylinker, theamplified region includes BglII site) and CMV-tt83-1R(ATTTGGAGTTGGCCTTAATATACTGGCCCATGGTATATCTCCTTCTTAAAGT) (SEQ ID NO: 12).For the second round, the PCR product from the first amplification wasdiluted 1:50 and re-amplified with primers pET-220 (SEQ ID NO: 11) andCMV-tt83Sal-R2 (GACGTCGACGCTCGGTAATCCCGATAAATTTGGAGTTGGCCTTAATATACTG)(SEQ ID NO: 13). The resulting PCR product (cDNA of SEQ ID NO: 14 codingfor CMV-Ntt830 of SEQ ID NO:6) was subcloned in BglII/SaLI sites ofpET-CMVwt. The correct clone was identified by sequencing and designatedpET-CMV-Ntt830.

Example 4 Expression of CMV-Ntt830 in E. coli Leading to Modified VLPsof CMV

To obtain CMV-Ntt830 VLPs, E. coli C2566 cells (New England Biolabs,Ipswich, USA) were transformed with the CMV-Ntt830 gene-containingplasmid pET-CMV-Ntt830. After selection of clones with the highestexpression levels of target protein, E. coli cultures were grown in 2×TYmedium containing kanamycin (25 mg/1) in a rotary shaker (200 rev/min;Infors, Bottmingen, Switzerland) at 30° C. to an OD600 of 0.8-1.0. The,cells were then induced with 0.2 mM IPTG, and the medium supplementedwith 5 mM MgCl₂. Incubation was continued on the rotary shaker at 20° C.for 18 h. The resulting biomass was collected by low-speedcentrifugation and frozen at −20° C. After thawing on ice, the cellswere suspended in buffer containing 50 mM sodium citrate, 5 mM sodiumborate, 5 mM EDTA, 5 mM mercaptoethanol (pH 9.0, buffer A) and disruptedby sonication. Insoluble proteins and cell debris were removed bycentrifugation (13,000 rpm, 30 min at 5° C.). The soluble CMV-Ntt830protein in clarified lysate was pelleted using saturated ammoniumsulfate (1:1, vol/vol) overnight at +4° C. Precipitated proteins weresolubilized in the buffer A (without mercaptoethanol) for 4 h at +4° C.Insoluble proteins were removed by low speed centrifugation (13,000 rpm,15 min at 4° C.). Soluble CMV-Ntt830-containing protein solution wasseparated from cellular proteins by ultracentrifugation (SW28 rotor,Beckman, Palo Alto, USA; at 25,000 rpm, 6 h, 5° C.) in a sucrosegradient (20-60% sucrose in buffer A, without mercaptoethanol,supplemented with 0.5% Triton X-100). The gradient was divided into sixfractions, starting at the bottom of the gradient. Fractions containingrecombinant CMV-Ntt830 were combined and dialyzed against 200 volumes of5 mM sodium borate, 2 mM EDTA (pH 9.0) to remove the sucrose and TritonX-100. After dialysis, CMV-Ntt830 solution was sterilized by filtrationthrough a 0.2μ filter. Next, CMV-Ntt830 was concentrated using Type70rotor (Beckman, Palo Alto, USA) ultracentrifugation through the 20%sucrose “cushion” under sterile conditions (50 000 rpm, 4 h, +5° C.).The concentration of purified CMV-Ntt830 was estimated using the QuBitfluorometer in accordance with manufacturer's recommendations(Invitrogen, Eugene, USA). Concentrated VLP solutions (approx. 3 mg/ml)were stored at +4° C. in 5 mM sodium borate, 2 mM EDTA, buffer (pH 9.0).All steps involved in the expression and purification of VLP weremonitored by SDS-PAGE using 12.5% gels. To demonstrate the presence ofthe tetanus toxoid epitope in CMV VLPs, mass spectrometric analysis ofthe purified CMV-Ntt830 VLPs was used. As shown in FIG. 3C, the majorpeak obtained corresponds to the theoretical molecular mass of theprotein if the first methionine is removed which occurs during proteinsynthesis in E. coli cells. Dynamic light scattering and electronmicroscopy confirmed isometric particle morphology similar to CMVwt VLPs(FIGS. 4A and 4B).

Example 5 Cloning of a Modified Coat Protein of CMV Containing a PADREEpitope (CMV-Npadr)

To introduce the PADRE epitope coding sequence in CMVwt gene, PCRmutagenesis was carried out using as the template for amplification andsubcloning the pET-CMVwt plasmid (see also Example 2 and 3). For theamplification following primers were used: pET-220 (SEQ ID NO: 11) andCMV-padrSal-R (GACGTCGACGCGCGGCCGCCTTGAGGGTCCACGC GGCCACAAATTTCGCCATGGT)(SEQ ID NO:15). The resulting PCR product (cDNA of SEQ ID NO:16 codingfor CMV-Npadr of SEQ ID NO:7) was again subcloned in BglII/SalI sites ofpET-CMVwt. The correct clone was identified by sequencing and designatedas pET-CMV-Npadr.

Example 6 Expression of CMV-Npadr in E. coli Leading to Modified VLPs ofCMV

The procedures for expression and purification of CMV-Npadr wereessentially the same as for CMV-Ntt830 and are described in Example 4.To demonstrate the presence of the PADRE epitope in CMV VLPs, the massspectrometric analysis of the purified CMV-Npadr VLPs was used. As shownin FIG. 3B, the major peak obtained corresponds to the theoreticalmolecular mass of the protein if the first methionine is removed whichoccurs during protein synthesis in E. coli cells. Dynamic lightscattering and electron microscopy analysis confirmed isometric particlemorphology, (FIG. 5A and FIG. 5B).

Example 7 Cloning of Fel d 1 Fusion Proteins

A Fel d1 fusion protein (named F12H6GGC) consisting of chain 1 of Fel d1fused to the N-terminus of chain 2 of Fel d1 via a 15 amino acidsequence (GGGGS)₃ (SEQ ID NO:17) and incorporating a HHHHHHGGC sequence(SEQ ID NO:18) fused to the C-terminus of chain 2 of Fel d1 was producedby oligonucleotide directed gene synthesis. The correspondingoligonucleotide sequence has the sequence of SEQ ID NO:19, wherein theprotein sequence of F12H6GGC has the sequence of SEQ ID NO:20:

MEICPAVKRDVDLFLTGTPDEYVEQVAQYKALPVVLENARILKNCVDAKMTEEDKENALSVLDKIYTSPLCGGGGSGGGGSGGGGSVKMAETCPIFYDVFFAVANGNELLLDLSLTKVNATEPERTAMKKIQDCYVENGLISRVLDGLVMTTISSSKDCMGEAVQNTVEDLKLNTLGRHHHHHHGGC

After synthesis of the gene, it was excised from its helper plasmid andsubcloned in frame into NdeI/XhoI sites of the plasmid pET42a(+)(Novagen, USA) resulting in the expression vector pET42-F12H6GGC.

Fel d1 fusion proteins with an additional glycine residue at theC-terminus (named F12H6GGCG) or without a hexa-histidine sequence (namedF12GGC) or without a hexa-histidine but with an additional glycineresidue at the C-terminus (named F12GGCG) were produced by PCRmutagenesis using the plasmid pET42-F12H6GGC as a template. Theoligonucleotide primers used in the PCRs to produce these fusionproteins were:

For F12H6GGCG, the forward primer was Fel_BglF (SEQ ID NO:21) and thereverse primer was Fel6H-cgR (SEQ ID NO:22).

For F12GGC, the forward primer was Fel_BglF (SEQ ID NO:21) and thereverse primer was Feld-dHR (SEQ ID NO:23).

For F12GGCG, the forward primer was Fel_BglF (SEQ ID NO:21) and thereverse primer was Feld-dH-cgR (SEQ ID NO:24).

All PCR products were cut with restriction enzymes BglII/XhoI andsubcloned back into vector pET42-F126HGGC at the same excision sites.After isolation of plasmid DNA, the introduced changes were confirmedusing a BigDye cycle sequencing kit and an ABI Prism 3100 Geneticanalyzer (Applied Biosystems, Carlsbad, USA). The resulting expressionvectors were named as pET42-F12H6GGCG, pET42-F12GGC and pET42-F12GGCG.They correspondingly encode the Fel d1 fusion proteins F12H6GGCG (SEQ IDNO: 25), F12GGC (SEQ ID NO: 26) and F12GGCG (SEQ ID NO:27).

(SEQ ID NO: 25) MEICPAVKRDVDLFLTGTPDEYVEQVAQYKALPVVLENARILKNCVDAKMTEEDKENALSVLDKIYTSPLCGGGGSGGGGSGGGGSVKMAETCPIFYDVFFAVANGNELLLDLSLTKVNATEPERTAMKKIQDCYVENGLISRVLDGLVMTTISSSKDCMGEAVQNTVEDLKLNTLGRHHHHHHGGCG  (SEQ ID NO: 26)MEICPAVKRDVDLFLTGTPDEYVEQVAQYKALPVVLENARILKNCVDAKMTEEDKENALSVLDKIYTSPLCGGGGSGGGGSGGGGSVKMAETCPIFYDVFFAVANGNELLLDLSLTKVNATEPERTAMKKIQDCYVENGLISRVLDGLVMTTISSSKDCMGEAVQNTVEDLKLNTLGRGGC (SEQ ID NO: 27)MEICPAVKRDVDLFLTGTPDEYVEQVAQYKALPVVLENARILKNCVDAKMTEEDKENALSVLDKIYTSPLCGGGGSGGGGSGGGGSVKMAETCPIFYDVFFAVANGNELLLDLSLTKVNATEPERTAMKKIQDCYVENGLISRVLDGLVMTTISSSKDCMGEAVQNTVEDLKLNTLGRGGCG

The hexa-histidine sequence enables purification by metal chelateaffinity chromatography and the C-terminal sequence comprising GGC orGGCG (SEQ ID NO:28) enables coupling of the Fel d1 fusion proteins toCMV-Ntt830 and CMV-Npadr.

Example 8 Expression and Purification of Fel d 1 Fusion Proteins

Expression of Fel d1 Fusion Proteins in E. coli.

The Fel d1-expression vectors pET42-F12H6GGC, pET42-F, 12H6GGCG,pET42-F12GGC and pET42-F12GGCG were transformed into E. coli C2566 cells(New England Biolabs, Ipswich, USA). Clones expressing the highestlevels of target protein were selected and used in further experiments.Expression of the various recombinant Fel d1 fusion proteins wasperformed in the following way. Cultures of E. coli harboring expressionplasmids were grown in 2×TY medium containing kanamycin (25 mg/1) on arotary shaker (200 rev/min; Infors, Bottmingen, Switzerland) at 30° C.to an OD600 of 0.8-1.0. Expression of the Fel d1 fusion protein geneswas then induced by adding 0.2 mM IPTG. The medium was supplemented with5 mM MgCl₂. Incubation was continued on a rotary shaker at 20° C. for 18h. The resulting biomass was collected by low-speed centrifugation andfrozen at −20° C. until purification.

Purification of Hexa-Histidine-Tagged Fel d1 Fusion Proteins.

For purification of F12H6GGC and F12H6GGCG fusion proteins, the USBPrepEase Kit (Affymetrix, High Wycombe, UK) was used according tomanufacturer's instructions. After thawing on ice, E. coli cells from100 ml culture (approx. 0.75 g) were suspended in 1×LEW buffercontaining 5 mM DTT and then disrupted by sonication. Insoluble proteinsand cell debris were removed by centrifugation (13,000 rpm, 30 min at 5°C.). The clarified lysate was applied to a Ni-IDA column, washed twicewith the same buffer (without DTT) and eluted with 2×1.5 ml of imidazolecontaining 1×E buffer. The fractions containing Fel d1 were identifiedby SDS/PAGE (FIG. 6A) and twice dialyzed against 200 volumes of thebuffer (20 mM sodium phosphate, 2 mM EDTA, pH 7.0). After dialysis, theprotein concentration was estimated using a QuBit fluorometer inaccordance with manufacturer's instructions (Invitrogen, Eugene, USA) orby UV spectrophotometric measurement at 280 nm. The identity of thepurified proteins was confirmed by mass spectrometric analysis (FIG. 6B)and by Western blot using anti-His-tag antibodies (Novagen, Cat. No.71840-3; data not shown).

Purification of Fel d1 Fusion Proteins without Hexa-Histidine Tags.

For purification of F12GGC and FG12GGCG fusion proteins, anion exchangeand hydrophobic interaction chromatography were used. Three grams ofIPTG induced E. coli were disrupted by sonication in 20 ml of lysisbuffer LB (20 mM Tris/HCl pH 8.0, 50 mM NaCl, 5 mM DTT). Aftersonication the solution was centrifuged for 15 min at 15 000 g and thesupernatant collected. Ammonium sulfate was added with constant stirringuntil 30% saturation was achieved then incubated for 5 min at RT. Aftercentrifugation, solid ammonium sulfate was added to the recoveredsupernatant until 50% saturation. After centrifugation, protein pelletswere collected and dissolved in 2 ml of LB and excess salt removed witha 5 ml HiTrap™ Desalting Column (GE Healthcare Life Sciences)equilibrated with LB. The desalted protein eluate was loaded onto a 1 mlHiTrap™ Capto™ DEAE column equilibrated with LB. Bound F12GGC orFG12GGCG were eluted with an increasing gradient of NaCl. Fractionscontaining Fel d1 fusion proteins were collected and pooled. Theresulting solution was diluted with 4 volumes of 20 mM Tris/HCl pH 8.0,5 mM DTT and loaded onto a MonoQ 5/50 GL column in LB and eluted with anincreasing NaCl gradient. Fractions containing Fel d1 fusion proteinswere collected and pooled. 5 M NaCl was added until a concentration of2.5 M was reached and DTT added to the solution, to maintain aconcentration of 5 mM. The Fel d1 containing solution was then loadedonto a 1 ml HiTrap™ Butyl HP column in 2.5 M NaCl, 5 mM DTT and elutedwith a continuously decreasing NaCl concentration. Fractions containingthe Fel d1 fusion proteins were collected and pooled. All purificationsteps were monitored by Coomassie-stained SDS/PAGE gels (FIG. 6C). Theidentity of purified proteins was confirmed by Western blot usingpolyclonal antibodies raised against recombinant Fel d1 (data notshown).

Example 9 Authenticity of Recombinant Fel d1 Fusion Protein(s)

Fel d1 Fusion Proteins are Similarly Recognized by Fel d1-SpecificMonoclonal Antibodies.

The binding of the Fel d1 fusion protein F12H6GGC and natural Fel d1(nFel d1) to Fel d1-specific monoclonal antibodies (mAb) was comparedusing a sandwich ELISA Fel d1 ELISA kit (6F9/3E4) from Indoorbiotechnologies (Cardiff, UK). To this end, Nunc ELISA plates werecoated with the anti-Fel d1 mAb 6F9 (at 1 microg/ml) at 4° C. overnight.Plates were washed with PBS containing 0.05% Tween 20 (PBST) and blockedwith Superblock (Invitrogen) for 2 h at room temperature (RT). NaturalFel d1 as well as F12H6GGC (1 μg/ml) were serially diluted 1:3 andincubated for 2 h at RT. Plates were washed with PBST and biotinylatedanti-Fel d1 mAb 3E4 (at 1 μg/ml) was added and incubated for 1 h at RT.Detection utilized Streptavidin conjugated to horse radish peroxidase(HRPO). To this end, plates were washed with PBST thenStreptavidin-Peroxidase (Sigma, 1:1000 dilution) was added to the platesfor 30 min at RT. Detection was performed with OPD substrate solutionand 5% H₂SO₄ as stop solution. The absorbance was measured using anELISA reader (BioRad) at 450 nm.

Natural Fel d1 and F12H6GGC gave similar titers in the ELISA whichdemonstrates they were similarly recognized by Fel d1-specific mAbs thusconfirming the authenticity of the recombinant Fel d1 F12H6GGC (FIG. 7).

Recombinant Fel d1 Fusion Proteins Activate Basophils in Whole Blood ofCat Allergic Patients.

Blood of cat allergic patients contain basophils which carry Feld1-specific IgE antibodies on their surface which, upon allergenexposure, crosslink the FcεRI and cause degranulation. To check theability of recombinant Fel d1 to cause degranulation, whole blood from aFel d1-allergic patients was collected and used in combination withrecombinant Fel d1 fusion protein F12H6GGC in a Basophil Activation Testkit of Bühlmann Laboratories (Flow Cast®, FK CCR). This assay measuresup-regulation of an exclusive degranulation marker CD63 on CCR3+basophils. Briefly, 100 μl of stimulation buffer was mixed with 50 μl ofEDTA-treated whole blood. In addition, 50 μl of various dilutions ofnatural Fel d1 or recombinant Fel d1 fusion protein F12H6GGC were added.Positive control solutions including a mAb against FcεRI as well as anunspecific cell activator (fMLP) were also tested in the assay. Stainingdye (20 μl per sample), containing anti-CCR3 Ab labeled to PE andanti-CD63 Ab labeled to FITC, was added and incubated at 37° C. for 25min. Erythrocytes were subsequently lysed adding lysis buffer. After 10min incubation, the samples were centrifuged at 500×g for 5 min andwashed with wash buffer (PBS containing 2% FCS). After a secondcentrifugation step, the cell pellets were suspended in 200 μl washbuffer and acquired using a flow cytometer (FACS Calibur). The sampleswere analyzed with Cell Quest Pro software. The percentage of the CD63expression on CCR3+ basophils was analyzed.

Recombinant Fel d1 fusion proteins was found to readily triggerdegranulation of basophils from cat allergic patients. Moreover, whencompared to natural Fel d1, similar levels of degranulation wereachieved thus demonstrating authenticity of the recombinantly producedFel d1 fusion proteins. (FIG. 8A/FIG. 8B).

Example 10 Coupling of Fel d1 Fusion Proteins to CMV-Ntt830 and CMV-VLPs

The Fel d1 fusion protein F12H6GGC was covalently linked to CMV-Ntt830and CMV-Npadr VLPs using the heterobifunctional chemical cross-linkersuccinimidyl-6-[(β-maleimidopropionamido) hexanoate] (SMPH) in thefollowing way.

CMV-Ntt830 and CMV-Npadr virus-like particles stored in 5 mM Na-borate,2 mM EDTA buffer, pH 9.0, were subject to buffer exchange with 20 mMNa-phosphate containing 30% sucrose and 2 mM EDTA using PD10 columns (GEHealthcare). A solution of CMV-Npadr or CMV-Ntt830 VLPs reacted for 60min at RT with 7.5× molar excess of heterobifunctional crosslinker SMPH.Unreacted SMPH was removed with PD10 columns in 20 mM Na-phosphatecontaining 30% sucrose and 2 mM EDTA.

Fel d1 fusion protein F12H6GGC was treated with 10× molar excess TCEP(Thermo Fisher). Derivatized CMV-Ntt830 and CMV-Npadr-VLPs were reactedwith 1× or 2× molar excess of recombinant Fel d1 fusion protein F12H6GGCfor 3 h at 23° C. The coupling reaction was analyzed by reducingSDS-PAGE (NuPAGE® 4-12% Bis-Tris gel) stained with Coomassie Blue.Protein bands with masses of approximately 44.5 kDa and 69 kDa wereevident after the chemical conjugation reaction (data not shown). Thesebands correspond to the CMV coat protein (24.5 kDa) covalently linkedwith the Fel d1 fusion protein F12H6GGC (20 kDa) and two CMV coatprotein molecules covalently linked with (49 kDa) one Fel d1 fusionprotein F12H6GGC respectively indicating the formation of Fel d1-CMVVLPs. Analogously, further Fel d1 fusion proteins such as the one of SEQID NO:25 were covalently linked to CMV-Ntt830 VLPs.

Example 11 Immune Response to Fel d1-CMV VLP in Mice

Groups of three female Balb/c mice were immunized with either Feld1-CMV-Ntt830-VLP, prepared as described in Example 10, orCMV-Ntt830-VLP simply mixed with Fel d1 fusion protein F12H6GGC. Bothcompositions contain the same amount of the Fel d1 fusion protein. 10 μgof each composition was prepared in 150 mM PBS, pH 7.4 and injected in avolume of 150 μl intravenously on day 0 and day 14. Mice were bled ondays 0 (pre-immune), day 14 and 21, and sera were analyzed by ELISA fornatural Fel d1 specific IgG-antibodies.

NUNC ELISA plates were coated with natural Fel d1 (IndoorBiotechnologies) in PBS with a concentration of 1 μg/ml overnight at 4°C. The plates were blocked with Superblock (Invitrogen). A serialdilution of the sera was performed in order to detect OD50. OD50describes the reciprocal of the dilution, which reaches half of themaximal OD value. IgG-antibodies specific for Fel d1 were detected withan anti-mouse IgG antibody directly labeled to horseradish dishperoxidase (HRPO) (Jackson). The conversion of o-phenylenediaminedihydrochloride (OPD) by the HRPO was measured as color reaction at 450nm, which was stopped by adding 5% sulfuric acid (H₂SO₄) after 7 minutesincubation.

After only a single immunization, Fel d1-specific IgG antibodies weredetected in mice (on day 14). The response was boosted by a secondinjection. Fel d1-CMV VLPs significantly increased the induction of Feld1 specific IgG antibodies compared to the mixed compositionsdemonstrating the immune-enhancing effect of chemical conjugation of theFel d1 fusion proteins to the VLP (FIG. 9).

Example 12 Immune Response to Fel d1-CMV VLPs in Cats

To investigate the immunogenicity and efficacy of Fel d1-CMV-Ntt830 VLPin the target species, female European shorthair cats were immunized 3×(at intervals of 21 days) via the intramuscular route (hind limb) with100 μg of Fel d1-CMV-Ntt830 VLP formulated in PBS either with adjuvant(15 μg Saponin Matrix M; n=3) or without adjuvant (n=3).

Blood was collected prior to immunization and on days, 22, 43, 58, 71and 85. After clotting and centrifugation serum samples were storedfrozen until assay. Saliva samples were collected from the animals byinserting a sterile swab into the mouth. This was performed prior toimmunization and on days 64 and 85.

A. Measurement of IgG Antibody Against (i) Fel d1 and (ii) CMV Carrierin Immunized Cats.

An ELISA assay was used to detect (i) Fel d1 and (ii) CMV specific IgGantibodies in sera from immunized cats. Briefly:

i) Natural Fel d1 (Indoor Biotechnologies), 1 μg/ml in PBS, was appliedovernight to NUNC ELISA plates which were then washed and blocked with2% BSA in PBS Tween 20 (0.05%). After washing, serially diluted cat serawere applied to the plates. After further washing, goat anti-cat IgGantibody labeled with horseradish peroxidase (HRPO) was applied to theplates. Following a final washing step, O-phenylenediaminedihydrochloride (OPD) was added and, after 7 minutes, the reaction wasstopped with 5% sulfuric acid. The conversion of OPD by HRPO wasmeasured at 450 nm. The titer is reported as OD50 which is thereciprocal of the dilution which reaches half of the maximal OD value.

Prior to immunization there was no significant anti-Fel d1 IgG. Feld1-specific IgG was detected on day 22 after a single immunization.After the second immunization on day 22, the response increased furtherand was maintained at high levels following the third injectionadministered on day 43. The antibody titers slowly declined thereafter.A similar result was obtained for cats, which had received the Feld1-CMV-Ntt830 VLP combined with adjuvant (FIG. 10A).

ii) Cucumber mosaic virus-like particle (CMVwt), 1 μg/ml in 0.1 M sodiumhydrogen carbonate (pH 9.6), was applied overnight to NUNC ELISA plates.After washing, the plates were blocked with 2% BSA in PBS Tween 20(0.05%). After washing, serially diluted cat sera were applied to theplates. After further washing, goat anti-cat IgG antibody labeled withHRPO was applied to the plates. Following a final washing step, OPD wasadded and, after 7 minutes, the reaction was stopped with 5% sulfuricacid. The conversion of OPD by HRPO was measured at 450 nm. The titer isreported as OD50 which is the reciprocal of the dilution which reacheshalf of the maximal OD value.

Prior to immunization there was no significant anti-CMV IgG.CMV-specific IgG was detected on day 22 after a single immunization.After the second immunization on day 22, the response increased furtherand was maintained at high levels following the third injection appliedon day 43. The antibody titer slowly declined thereafter. A similarresult was obtained for cats, which had received the Fel d1-CMV-Ntt830VLP with adjuvant (FIG. 10B).

B. Determination of Fel d1 and CMV-VLP Specific Antibodies in SalivaCollected from Immunized Cats.

One ml of PBST was pipetted onto the cotton swabs (used to collectsaliva) which were incubated at RT for 30 min at 50 rpm on a rotarymixer. The liquid was separated from the swab by centrifugation at 4000rpm for 10 min using a sieve (cell strainer, BD #352350) in a 50 mlFalcon tube. The flow through was collected and used for ELISA.

To detect anti-Fel d1 IgG and IgA antibodies an indirect ELISA methodwas used. Briefly, 1 μg/ml of natural Fel d1 in PBS, was appliedovernight at 4° C. to NUNC ELISA plates which were then washed andblocked with 2% BSA in PBS Tween 20 (0.05%). After washing, seriallydiluted (1:3) saliva extracts were applied to the plates which weresubsequently washed. For detection of IgG antibodies, goat anti-cat IgGantibody labeled with HRPO was applied. Alternatively, for detection ofIgA antibodies, goat anti-cat IgA antibody labeled with HRPO was addedto the plates. Following a final washing step, OPD was added and, after7 minutes, the reaction was stopped with 5% sulfuric acid. Theconversion of OPD by HRPO was measured at 450 nm.

Salivary CMV-specific antibodies were similarly measured usingrecombinantly expressed CMVwt VLP coated ELISA plates.

Following the immunization, Fel d1-specific IgG antibodies (FIG. 11A),above the individual pre-immunization base-line levels, were measured onday 64 from five cats and on day 85 from all six cats. Fel d1-specificIgA antibodies (FIG. 11B), above the individual pre-immunizationbase-line levels, were measured on day 64 from five cats and on day 85from five cats. CMV-specific IgG antibodies (FIG. 11C) abovepre-immunization base-line levels were measured on day 64 from five catsand on day 85 from five cats. CMV-specific IgA antibodies (FIG. 11D)were measured on day 64 from all six cats and on day 85 from all sixcats.

C. Determination of Immune Complexes Consisting of Endogenous Fel d1 andAnti-Fel d1 IgA Antibodies in Saliva Collected from Immunized Cats.

A mixture of three different mAbs (5 μg/ml in PBS), specific for threenon-overlapping Fel d1 epitopes, was coated onto NUNC ELISA platesovernight at 4° C. Plates were washed and blocked (2% BSA/PBST) for 2 hat RT. Neat and serially diluted saliva (1:3) extracts were applied tothe plates. Immune-complexes comprising endogenous Fel d1 and IgAantibodies were detected with a goat anti-cat IgA Ab-HRPO from AbDSerotec. Following a final washing step, OPD was added and, after 7minutes, the reaction was stopped with 5% sulfuric acid. The conversionof OPD by HRPO was measured at 450 nm.

Immune complexes consisting of endogenous Fel d1 and IgA antibodiesabove pre-immunization base-line levels were detected in all cats eitheron d64 or on d85 (FIG. 12).

Example 13 Saliva Samples from Fel d1-CMV-Ntt830 VLP Immunized Cats ShowReduced Degranulation of Basophils from Cat Allergic Patient

The ability of immunization with Fel d1-CMV-Ntt830 VLP to inhibitsalivary Fel d1 mediated basophil degranulation was determined using theBasophil activation test as described in Example 9. To this end, salivasamples from cats before and after immunization were collected andextracted as described in Example 12. The Basophil activation test wasperformed using 50 μl of anti-FcεRI mAb as positive control or 50 μlsaliva samples from cats before and after immunization.

Briefly, 100 μl of stimulation buffer was mixed with 50 μl ofEDTA-treated whole blood. In addition, 50 μl saliva samples from catsbefore and after immunization (day 85) or or a mAb against FcεRI as apositive control were added. Staining dye (20 μl per sample), containinganti-CCR3 Ab labeled to PE and anti-CD63 Ab labeled to FITC, was addedand incubated at 37° C. for 25 min. Erythrocytes were subsequently lysedadding lysis buffer. After 10 min incubation, the samples werecentrifuged at 500×g for 5 min and washed with wash buffer (PBScontaining 2% FCS). After a second centrifugation step, the cell pelletswere suspended in 200 μl wash buffer and acquired using a flow cytometer(FACS Calibur). The samples were analyzed with Cell Quest Pro software.The percentage of the CD63 expression on CCR3+ basophils was analyzed.

Saliva extracts from 5 of 6 cats taken after immunization on day 85showed decreased levels of degranulation by up to 20% when compared tosaliva extracts before immunization (FIG. 13). When extrapolated to atitration curve constructed with natural Fel d1 in said Basophilactivation test and said cat allergic patient, a reduction of 20% indegranulation corresponds to a 13-fold decrease in Fel d1 concentration.This indicates that a significant reduction in allergenic Fel d1 insaliva was achieved.

Example 14 Effect of Cat Immunization Assessed by a Clinical Trial withCat Allergic Subject

A titrated skin prick test of cat allergic human subjects was used tocompare the allergenicity of cat fur extracts obtained before and afterimmunization of cats with Fel d1-CMV-Ntt830 VLP.

Preparation of Cat Fur Extract

Three female European short haired cats were immunized four timessubcutaneously with 100 ug of Fel d1-CMV-Ntt830 VLP (prepared asdescribed in Example 10 comprising SEQ ID NO:25) on days 1, 22, 43 and256. Fur samples from cats were obtained by brushing cats prior toimmunization on day 1 and after the fourth immunization on day 312.Collected fur samples were stored frozen until preparation.

In order to prepare fur extracts, 0.03 g of fur were transferred into anextraction vial (1.5 ml tube) and 1 ml of phosphate buffered salinecontaining 0.05% Tween20 was added to the vial. The extraction tubeswere placed into a thermoshaker with a set temperature of 23° C. toincubate at 550 rpm for 1.5 hours. After incubation, the extractiontubes were transferred to a table top Eppendorf centrifuge and spun for10 min at RT at 16.000×g. The supernatants were transferred into clean1.5 ml tubes and stored frozen until analysis.

Skin Prick Test

Frozen cat fur extracts (75 μl solution in 0:5 ml Eppendorf tube) werethawed shortly before use and diluted in three-fold serial dilutionswith PBS-Tween 20.

The positive allergic statuses of the cat allergic subjects wereconfirmed using a conventional skin prick test on the lateral side ofthe left volar forearm with a cat fur extract.

The skin prick test used for patient screening and assessment of cat furextracts is described briefly. A suitable area of skin was selected fortesting, on the volar aspect of the forearms. The skin was clean, dryand free from fat, creams or cosmetics by using rubbing alcohol providedby the cantonal pharmacy. Eczematous or inflamed areas of skin wereavoided. The prick sites were marked and numbered using a skin markingpen. The cubital area and wrist were avoided. The vertical andhorizontal distance between two allergen extracts was at least 2 cm toavoid cross contamination. A droplet (10 μl) of the cat fur solution wasbrought onto the skin at the appropriate position utilizing a 20 μlGilson pipette. The skin prick needle was then pressed through thedroplet, pricking the allergen into the dermis. The pressure was appliedfor approximately one second and the strength of the pressure was soughtto be the same for all applications. After 15 minutes the contours ofeach wheal were encircled using a skin marking pen. These lines weredrawn on the red skin surrounding the wheal, without crossing orcovering any part of it. It was ensured that no part of the red skinsurrounding the wheal appeared inside the encircled part. A copy of theencircled mark was obtained by sticking a transparent self-adhesive tapeonto the wheal and then sticking it onto paper to keep a permanentrecord. The area of the wheal size (mm2) was calculated.

At each patient visit, diluted fur extracts from one cat were tested.Cat fur extracts taken before the immunization was tested on the rightarm, cat fur solution of fur taken after the immunization was tested onthe left arm.

Results

Seven cat allergic patients (aged 18-65 years, male and female) wereincluded in the single center, open-label clinical study. A comparisonof wheal sizes from 16 skin-prick tests (out of a possible 21) thatcompared pre-immunization and post-immunization fur extracts weresuccessfully obtained.

An analysis of the mean wheal sizes from these 16 skin prick tests showsthat fur extracts obtained from immunized cats induced smaller whealsizes than those collected prior to immunization (FIG. 14). This datasuggests cat fur extracts were less allergenic after immunization withFel d1-CMV-Ntt830 VLP.

The invention claimed is:
 1. A method of reducing the allergenicity of acat for a human exposed to fur of said cat, wherein said methodcomprises administering an effective amount of a composition to saidcat, and wherein said composition comprises (i) a virus-like particle(VLP) with at least one first attachment site; (ii) at least one Fel d1protein with at least one second attachment site; and wherein saidvirus-like particle and said Fel d1 protein are linked through said atleast one first and said at least one second attachment site whereinsaid reducing the allergenicity of said cat for said human exposed tosaid cat is (i) reducing the level or severity of the allergic responsegenerated by said human, or (ii) reducing at least one allergic symptomof said human.
 2. The method of claim 1, wherein said reducing theallergenicity of said cat is effected by generating immune complexesformed of Fel d1 and Fel d1-antibodies in the fur of said cat.
 3. Themethod of claim 1 wherein (i) said reduction in the level or severity ofthe allergic response generated by said human, or (ii) said reduction ofsaid at least one allergic symptom of said human, is expressed by a lesspositive skin prick test, nasal provocation test or conjunctivalprovocation test.
 4. The method of claim 1, wherein said VLP is amodified VLP comprising at least one modified VLP polypeptide, whereinsaid modified VLP polypeptide comprises (a) a VLP polypeptide, and (b) aT helper cell epitope, wherein said VLP polypeptide comprises (i) anamino acid sequence of a coat protein of a virus; or (ii) an amino acidhaving a sequence identity of at least 90% to said coat protein of avirus.
 5. The method of claim 1, wherein said VLP is a modified VLP ofcucumber mosaic virus (CMV), wherein said modified VLP of CMV comprisesat least one modified CMV polypeptide, wherein said modified CMVpolypeptide comprises (a) a CMV polypeptide, and (b) a T helper cellepitope; and wherein said CMV polypeptide comprises (ii) an amino acidsequence of a coat protein of CMV; or (ii) an amino acid sequence havinga sequence identity of at least 90% to said coat protein of CMV.
 6. Themethod of claim 5, wherein said CMV polypeptide comprises (a) an aminoacid sequence of a coat protein of CMV, wherein said amino acid sequencecomprises SEQ ID NO:1 or (b) an amino acid sequence having a sequenceidentity of at least 90% of SEQ ID NO:1; and wherein said amino sequenceas defined in (a) or (b) in this claim comprises SEQ ID NO:34; orwherein said amino sequence as defined in (a) or (b) in this claimcomprises an amino acid sequence region, wherein said amino acidsequence region has a sequence identity of at least 90% with SEQ IDNO:34.
 7. The method of claim 5, wherein said T helper cell epitopereplaces a N-terminal region of said CMV polypeptide, and wherein saidN-terminal region of said CMV polypeptide comprises amino acids 2-12 ofSEQ ID NO:1.
 8. The method of claim 5, wherein said T helper cellepitope is a PADRE sequence, and wherein said T helper cell epitopecomprises the amino acid sequence of SEQ ID NO:5; or wherein said Thelper cell epitope is derived from tetanus toxin, and wherein said Thelper cell epitope comprises the amino acid sequence of SEQ ID NO:4. 9.The method of claim 5, wherein said CMV polypeptide comprises an aminoacid sequence of a coat protein of CMV, wherein said amino acid sequencecomprises SEQ ID NO:1 or an amino acid sequence having a sequenceidentity of at least 95% of SEQ ID NO:1; and wherein said amino sequencecomprises SEQ ID NO:34, and wherein said T helper cell epitope replacesthe N-terminal region of said CMV polypeptide, and wherein said replacedN-terminal region of said CMV polypeptide consists of 11 to 13consecutive amino acids.
 10. The method of claim 5, wherein saidmodified CMV polypeptide comprises an amino acid sequence of SEQ ID NO:6or SEQ ID NO:7.
 11. The method of claim 1, wherein said Fel d1 proteinis a Fel d1 fusion protein comprising chain 1 of Fel d1 and chain 2 ofFel d1, wherein chain 1 of Fel d1 and chain 2 of Fel d1 are fused eitherdirectly via one peptide bond or via a spacer, which links theN-terminus of one chain with the C-terminus of another chain.
 12. Themethod of claim 1, wherein said Fel d1 protein comprises an amino acidsequence selected from: (a) SEQ ID NO:20; (b) SEQ ID NO:25; (c) SEQ IDNO:26; (d) SEQ ID NO:27; or (e) SEQ ID NO:29.
 13. The method of claim 2,wherein said administration of said composition leads to said generatingof said immune complexes in the saliva, fur, skin or tears of said cat.14. The method of claim 3, wherein the fur from said cat before andafter said administration is used for said skin prick test, nasalprovocation test or conjunctival provocation test.
 15. The method ofclaim 4, wherein said VLP polypeptide comprises an amino acid sequenceof a coat protein of a plant virus.
 16. The method of claim 9, whereinsaid replaced N-terminal region of said CMV polypeptide consists of 11consecutive amino acids.
 17. The method of claim 16, wherein saidN-terminal region of said CMV polypeptide comprises amino acids 2-12 ofSEQ ID NO:1.
 18. The method of claim 5, wherein said modified CMVpolypeptide comprises an amino acid sequence of SEQ ID NO:6.
 19. Themethod of claim 18, wherein said Fel d1 protein comprises an amino acidsequence of SEQ ID NO:25.
 20. The method of claim 18, wherein said Feld1 protein comprises an amino acid sequence of SEQ ID NO:27.